Mammalian Cytokine; Related Reagents

ABSTRACT

Purified genes encoding cytokine from a mammal, reagents related thereto including purified proteins, specific antibodies, and nucleic acids encoding this molecule are provided. Methods of using said reagents and diagnostic kits are also provided.

This application is a conversion of provisional U.S. Patent ApplicationU.S. Ser. No. 60/053,765, filed Jul. 25, 1997, which is incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention pertains to compositions related to proteins whichfunction in controlling biology and physiology of mammalian cells, e.g.,cells of a mammalian immune system. In particular, it provides purifiedgenes, proteins, antibodies, and related reagents useful, e.g., toregulate activation, development, differentiation, and function ofvarious cell types, including hematopoietic cells.

BACKGROUND OF THE INVENTION

Recombinant DNA technology refers generally to the technique ofintegrating genetic information from a donor source into vectors forsubsequent processing, such as through introduction into a host, wherebythe transferred genetic information is copied and/or expressed in thenew environment. Commonly, the genetic information exists in the form ofcomplementary DNA (cDNA) derived from messenger RNA (mRNA) coding for adesired protein product. The carrier is frequently a plasmid having thecapacity to incorporate cDNA for later replication in a host and, insome cases, actually to control expression of the cDNA and therebydirect synthesis of the encoded product in the host.

For some time, it has been known that the mammalian immune response isbased on a series of complex cellular interactions, called the “immunenetwork”. Recent research has provided new insights into the innerworkings of this network. While it remains clear that much of theresponse does, in fact, revolve around the network-like interactions oflymphocytes, macrophages, granulocytes, and other cells, immunologistsnow generally hold the opinion that soluble proteins, known aslymphokines, cytokines, or monokines, play a critical role incontrolling these cellular interactions. Thus, there is considerableinterest in the isolation, characterization, and mechanisms of action ofcell modulatory factors, an understanding of which will lead tosignificant advancements in the diagnosis and therapy of numerousmedical abnormalities, e.g., immune system disorders. Some of thesefactors are hematopoietic growth factors, e.g., granulocyte colonystimulating factor (G-CSF). See, e.g., Thomson (1994; ed.) The CytokineHandbook (2d ed.) Academic Press, San Diego; Metcalf and Nicola (1995)The Hematopoietic Colony Stimulating Factors Cambridge University Press;and Aggarwal and Gutterman (1991) Human Cytokines Blackwell Pub.

Lymphokines apparently mediate cellular activities in a variety of ways.They have been shown to support the proliferation, growth, anddifferentiation of pluripotential hematopoietic stem cells into vastnumbers of progenitors comprising diverse cellular lineages making up acomplex immune system. Proper and balanced interactions between thecellular components are necessary for a healthy immune response. Thedifferent cellular lineages often respond in a different manner whenlymphokines are administered in conjunction with other agents.

Cell lineages especially important to the immune response include twoclasses of lymphocytes: B-cells, which can produce and secreteimmunoglobulins (proteins with the capability of recognizing and bindingto foreign matter to effect its removal), and T-cells of various subsetsthat secrete lymphokines and induce or suppress the B-cells and variousother cells (including other T-cells) making up the immune network.These lymphocytes interact with many other cell types.

Another important cell lineage is the mast cell (which has not beenpositively identified in all mammalian species), which is agranule-containing connective tissue cell located proximal tocapillaries throughout the body. These cells are found in especiallyhigh concentrations in the lungs, skin, and gastrointestinal andgenitourinary tracts. Mast cells play a central role in allergy-relateddisorders, particularly anaphylaxis as follows: when selected antigenscrosslink one class of immunoglobulins bound to receptors on the mastcell surface, the mast cell degranulates and releases mediators, e.g.,histamine, serotonin, heparin, and prostaglandins, which cause allergicreactions, e.g., anaphylaxis.

Research to better understand and treat various immune disorders hasbeen hampered by the general inability to maintain cells of the immunesystem in vitro. Immunologists have discovered that culturing thesecells can be accomplished through the use of T-cell and other cellsupernatants, which contain various growth factors, including many ofthe lymphokines.

From the foregoing, it is evident that the discovery and development ofnew lymphokines, e.g., related to G-CSF and/or IL-6, could contribute tonew therapies for a wide range of degenerative or abnormal conditionswhich directly or indirectly involve the immune system and/orhematopoietic cells. In particular, the discovery and development oflymphokines which enhance or potentiate the beneficial activities ofknown lymphokines would be highly advantageous. The present inventionprovides new interleukin compositions and related compounds, and methodsfor their use.

SUMMARY OF THE INVENTION

The present invention is directed to mammalian, e.g., rodent, canine,feline, primate, interleukin-B30 (IL-B30) and its biological activities.It includes nucleic acids coding for polypeptides themselves and methodsfor their production and use. The nucleic acids of the invention arecharacterized, in part, by their homology to cloned complementary DNA(cDNA) sequences enclosed herein, and/or by functional assays for growthfactor- or cytokine-like activities, e.g., G-CSF (see Nagata (1994) inThomson The Cytokine Handbook 2d ed., Academic Press, San Diego) and/orIL-6 (see Hirano (1994) in Thomson The Cytokine Handbook 2d ed.,Academic Press, San Diego), applied to the polypeptides, which aretypically encoded by these nucleic acids. Methods for modulating orintervening in the control of a growth factor dependent physiology or animmune response are provided.

The present invention is based, in part, upon the discovery of a newcytokine sequence exhibiting significant sequence and structuralsimilarity to G-CSF and IL-6. In particular, it provides primate, e.g.,human, gene encoding a protein whose mature size is about 168 aminoacids, and pig and murine, e.g., mouse, sequences. Functionalequivalents exhibiting significant sequence homology will be availablefrom other mammalian, e.g., cow, horse, and rat, and non-mammalianspecies.

In various protein embodiments, the invention provides: a substantiallypure or recombinant IL-B30 protein or peptide exhibiting at least about85% sequence identity over a length of at least about 12 amino acids toSEQ ID NO: 2; a natural sequence IL-B30 of SEQ ID NO: 2; and a fusionprotein comprising IL-B30 sequence. In certain embodiments, the homologyis at least about 90% identity and the portion is at least about 9 aminoacids; the homology is at least about 80% identity and the portion is atleast about 17 amino acids; or the homology is at least about 70%identity and the portion is at least about 25 amino acids. In otherembodiments, the IL-B30: comprises a mature sequence of Table 1; orexhibits a post-translational modification pattern distinct from naturalIL-B30; or the protein or peptide: is from a warm blooded animalselected from a mammal, including a primate; comprises at least onepolypeptide segment of SEQ ID NO: 2; exhibits a plurality of portionsexhibiting the identity; is a natural allelic variant of IL-B30; has alength at least about 30 amino acids; exhibits at least twonon-overlapping epitopes which are specific for a mammalian IL-B30;exhibits a sequence identity at least about 90% over a length of atleast about 20 amino acids to mammalian IL-B30; is glycosylated; has amolecular weight of at least 10 kD with natural glycosylation; is asynthetic polypeptide; is attached to a solid substrate; is conjugatedto another chemical moiety; is a 5-fold or less substitution fromnatural sequence; or is a deletion or insertion variant from a naturalsequence. Preferred embodiments include a composition comprising: asterile IL-B30 protein or peptide; or the IL-B30 protein or peptide anda carrier, wherein the carrier is: an aqueous compound, including water,saline, and/or buffer; and/or formulated for oral, rectal, nasal,topical, or parenteral administration. In fusion protein embodiments,the protein can have: mature protein sequence of Table 1; a detection orpurification tag, including a FLAG, His6, or Ig sequence; and/orsequence of another cytokine or chemokine.

Kit embodiments include those with an IL-B30 protein or polypeptide,and: a compartment comprising the protein or polypeptide; and/orinstructions for use or disposal of reagents in the kit.

In binding compound embodiments, the compound may have an antigenbinding site from an antibody, which specifically binds to a naturalIL-B30 protein, wherein: the IL-B30 is a mammalian protein; the bindingcompound is an Fv, Fab, or Fab2 fragment; the binding compound isconjugated to another chemical moiety; or the antibody: is raisedagainst a peptide sequence of a mature polypeptide of Table 1; is raisedagainst a mature IL-B30; is raised to a purified rodent IL-B30; isimmunoselected; is a polyclonal antibody; binds to a denatured IL-B30;exhibits a Kd of at least 30 μM; is attached to a solid substrate,including a bead or plastic membrane; is in a sterile composition; or isdetectably labeled, including a radioactive or fluorescent label. Kitscontaining binding compounds include those with: a compartmentcomprising the binding compound; and/or instructions for use or disposalof reagents in the kit. Often the kit is capable of making a qualitativeor quantitative analysis. Preferred compositions will comprise: asterile binding compound; or the binding compound and a carrier, whereinthe carrier is: an aqueous compound, including water, saline, and/orbuffer; and/or formulated for oral, rectal, nasal, topical, orparenteral administration.

Nucleic acid embodiments include an isolated or recombinant nucleic acidencoding an IL-B30 protein or peptide or fusion protein, wherein: theIL-B30 is from a mammal; and/or the nucleic acid: encodes an antigenicpeptide sequence of Table 1; encodes a plurality of antigenic peptidesequences of Table 1; exhibits at least about 80% identity to a naturalcDNA encoding the segment; is an expression vector; further comprises anorigin of replication; is from a natural source; comprises a detectablelabel; comprises synthetic nucleotide sequence; is less than 6 kb,preferably less than 3 kb; is from a mammal, including a primate;comprises a natural full length coding sequence; is a hybridizationprobe for a gene encoding the IL-B30; or is a PCR primer, PCR product,or mutagenesis primer. The invention also provides a cell, tissue, ororgan comprising such a recombinant nucleic acid, and preferably thecell will be: a prokaryotic cell; a eukaryotic cell; a bacterial cell; ayeast cell; an insect cell; a mammalian cell; a mouse cell; a primatecell; or a human cell.

Kit embodiments include those with such nucleic acids, and: acompartment comprising the nucleic acid; a compartment furthercomprising the IL-B30 protein or polypeptide; and/or instructions foruse or disposal of reagents in the kit. Typically, the kit is capable ofmaking a qualitative or quantitative analysis.

In certain embodiments, the nucleic acid: hybridizes under washconditions of 30° C. and less than 2M salt, or of 45° C. and/or 500 mMsalt, or 55° C. and/or 150 mM salt, to SEQ ID NO: 1; or exhibits atleast about 85% identity and/or the stretch is at least about 30nucleotides, or exhibits at least 90% identity and/or the stretch is atleast 55 nucleotides, or exhibits at least 95% and/or the stretch is atleast 75 nucleotides, to a primate IL-B30.

The invention embraces a method of modulating physiology or developmentof a cell or tissue culture cells comprising contacting the cell with anagonist or antagonist of a mammalian IL-B30. The method may be where:the contacting is in combination with an agonist or antagonist of G-CSFand/or IL-6; or the contacting is with an antagonist, including abinding composition comprising an antibody binding site whichspecifically binds an IL-B30.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

All references cited herein are incorporated herein by reference to thesame extent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

Outline

I. General

II. Purified IL-B30

-   -   A. physical properties    -   B. biological properties

III. Physical Variants

-   -   A. sequence variants, fragments    -   B. post-translational variants        -   1. glycosylation        -   2. others

IV. Functional Variants

-   -   A. analogs, fragments        -   1. agonists        -   2. antagonists    -   B. mimetics        -   1. protein        -   2. chemicals    -   C. species variants

V. Antibodies

-   -   A. polyclonal    -   B. monoclonal    -   C. fragments, binding compositions

VI. Nucleic Acids

-   -   A. natural isolates; methods    -   B. synthetic genes    -   C. methods to isolate

VII. Making IL-B30, mimetics

-   -   A. recombinant methods    -   B. synthetic methods    -   C. natural purification

VIII. Uses

-   -   A. diagnostic    -   B. therapeutic

IX. Kits

-   -   A. nucleic acid reagents    -   B. protein reagents    -   C. antibody reagents

X. Isolating receptors for IL-B30

I. General

The present invention provides amino acid sequences and DNA sequencesencoding various mammalian proteins which are cytokines, e.g., which aresecreted molecules which can mediate a signal between immune or othercells. See, e.g., Paul (1994) Fundamental Immunology (3d ed.) RavenPress, N.Y. The full length cytokines, and fragments, or antagonistswill be useful in physiological modulation of cells expressing areceptor. It is likely that IL-B30 has either stimulatory or inhibitoryeffects on hematopoietic cells, including, e.g., lymphoid cells, such asT-cells, B-cells, natural killer (NK) cells, macrophages, dendriticcells, hematopoietic progenitors, etc. The proteins will also be usefulas antigens, e.g., immunogens, for raising antibodies to variousepitopes on the protein, both linear and conformational epitopes.

A cDNA encoding IL-B30 was identified from a human cell line. Themolecule was designated huIL-B30. A related gene corresponding to a pigsequence was also identified. A rodent sequence, e.g., from mouse, isalso described.

The human gene encodes a small soluble cytokine-like protein, of about168 amino acids. The signal sequence probably is about 21 residues, andwould run from the Met to about Ala. See Table 1 and SEQ. ID. NO: 1 and2. IL-B30 exhibits structural motifs characteristic of a member of thelong chain cytokines. Compare, e.g., IL-B30, G-CSF, and IL-6, sequencesavailable from GenBank. See also Table 2.

TABLE 1 Nucleic acid (SEQ ID NO: 1) encoding IL-B30 from a primate,e.g., human. Translated amino acid sequence is SEQ ID NO: 2. ATG CTG GGGAGC AGA GCT GTA ATG CTG CTG TTG CTG CTG CCC TGG ACA 48 Met Leu Gly SerArg Ala Val Met Leu Leu Leu Leu Leu Pro Trp Thr −21−20                 −15                 −10 GCT CAG GGC AGA GCT GTG CCTGGG GGC AGC AGC CCT GCC TGG ACT CAG 96 Ala Gln Gly Arg Ala Val Pro GlyGly Ser Ser Pro Ala Trp Thr Gln −5                   1               5                  10 TGC CAG CAGCTT TCA CAG AAG CTC TGC ACA CTG GCC TGG AGT GCA CAT 144 Cys Gln Gln LeuSer Gln Lys Leu Cys Thr Leu Ala Trp Ser Ala His             15                  20                  25 CCA CTA GTG GGACAC ATG GAT CTA AGA GAA GAG GGA GAT GAA GAG ACT 192 Pro Leu Val Gly HisMet Asp Leu Arg Glu Glu Gly Asp Glu Glu Thr         30                  35                  40 ACA AAT GAT GTT CCCCAT ATC CAG TGT GGA GAT GGC TGT GAC CCC CAA 240 Thr Asn Asp Val Pro HisIle Gln Cys Gly Asp Gly Cys Asp Pro Gln     45                  50                  55 GGA CTC AGG GAC AAC AGTCAG TTC TGC TTG CAA AGG ATC CAC CAG GGT 288 Gly Leu Arg Asp Asn Ser GlnPhe Cys Leu Gln Arg Ile His Gln Gly 60                  65                  70                  75 CTG ATTTTT TAT GAG AAG CTG CTA GGA TCG GAT ATT TTC ACA GGG GAG 336 Leu Ile PheTyr Glu Lys Leu Leu Gly Ser Asp Ile Phe Thr Gly Glu                 80                  85                  90 CCT TCT CTGCTC CCT GAT AGC CCT GTG GCG CAG CTT CAT GCC TCC CTA 384 Pro Ser Leu LeuPro Asp Ser Pro Val Ala Gln Leu His Ala Ser Leu             95                 100                 105 CTG GGC CTC AGCCAA CTC CTG CAG CCT GAG GGT CAC CAC TGG GAG ACT 432 Leu Gly Leu Ser GlnLeu Leu Gln Pro Glu Gly His His Trp Glu Thr        110                 115                 120 CAG CAG ATT CCA AGCCTC AGT CCC AGC CAG CCA TGG CAG CGT CTC CTT 480 Gln Gln Ile Pro Ser LeuSer Pro Ser Gln Pro Trp Gln Arg Leu Leu    125                 130                 135 CTC CGC TTC AAA ATC CTTCGC AGC CTC CAG GCC TTT GTG GCT GTA GCC 528 Leu Arg Phe Lys Ile Leu ArgSer Leu Gln Ala Phe Val Ala Val Ala140                 145                 150                 155 GCC CGGGTC TTT GCC CAT GGA GCA GCA ACC CTG AGT CCC TAA 570 Ala Arg Val Phe AlaHis Gly Ala Ala Thr Leu Ser Pro                 160                 165coding sequence: ATGCTGGGGA GCAGAGCTGT AATGCTGCTG TTGCTGCTGC CCTGGACAGCTCAGGGCAGA GCTGTGCCTG GGGGCAGCAG CCCTGCCTGG ACTCAGTGCC AGCAGCTTTCACAGAAGCTC TGCACACTGG CCTGGAGTGC ACATCCACTA GTGGGACACA TGGATCTAAGAGAAGAGGGA GATGAAGAGA CTACAAATGA TGTTCCCCAT ATCCAGTGTG GAGATGGCTGTGACCCCCAA GGACTCAGGG ACAACAGTCA GTTCTGCTTG CAAAGGATCC ACCAGGGTCTGATTTTTTAT GAGAAGCTGC TAGGATCGGA TATTTTCACA GGGGAGCCTT CTCTGCTCCCTGATAGCCCT GTGGCGCAGC TTCATGCCTC CCTACTGGGC CTCAGCCAAC TCCTGCAGCCTGAGGGTCAC CACTGGGAGA CTCAGCAGAT TCCAAGCCTC AGTCCCAGCC AGCCATGGCAGCGTCTCCTT CTCCGCTTCA AAATCCTTCG CAGCCTCCAG GCCTTTGTGG CTGTAGCCGCCCGGGTCTTT GCCCATGGAG CAGCAACCCT GAGTCCCTAA Rodent, e.g., mouse, IL-B30(SEQ ID NO: 3 and 4): CGCTTAGAAG TCGGACTACA GAGTTAGACT CAGAACCAAAGGAGGTGGAT AGGGGGTCCA 60 CAGGCCTGGT GCAGATCACA GAGCCAGCCA GATCTGAGAAGCAGGGAACA AG ATG 115                                                          Met                                                          −21 CTG GATTGC AGA GCA GTA ATA ATG CTA TGG CTG TTG CCC TGG GTC ACT 163 Leu Asp CysArg Ala Val Ile Met Leu Trp Leu Leu Pro Trp Val Thr−20                 −15                 −10                  −5 CAG GGCCTG GCT GTG CCT AGG AGT AGC AGT CCT GAC TGG GCT CAG TGC 211 Gln Gly LeuAla Val Pro Arg Ser Ser Ser Pro Asp Trp Ala Gln Cys                  1               5                  10 CAG CAG CTC TCTCGG AAT CTC TGC ATG CTA GCC TGG AAC GCA CAT GCA 259 Gln Gln Leu Ser ArgAsn Leu Cys Met Leu Ala Trp Asn Ala His Ala         15                  20                  25 CCA GCG GGA CAT ATGAAT CTA CTA AGA GAA GAA GAG GAT GAA GAG ACT 307 Pro Ala Gly His Met AsnLeu Leu Arg Glu Glu Glu Asp Glu Glu Thr     30                  35                  40 AAA AAT AAT GTG CCC CGTATC CAG TGT GAA GAT GGT TGT GAC CCA CAA 355 Lys Asn Asn Val Pro Arg IleGln Cys Glu Asp Gly Cys Asp Pro Gln 45                  50                  55                  60 GGA CTCAAG GAC AAC AGC CAG TTC TGC TTG CAA AGG ATC CGC CAA GGT 403 Gly Leu LysAsp Asn Ser Gln Phe Cys Leu Gln Arg Ile Arg Gln Gly                 65                  70                  75 CTG GCT TTTTAT AAG CAC CTG CTT GAC TCT GAC ATC TTC AAA GGG GAG 451 Leu Ala Phe TyrLys His Leu Leu Asp Ser Asp Ile Phe Lys Gly Glu             80                  85                  90 CCT GCT CTA CTCCCT GAT AGC CCC ATG GAG CAA CTT CAC ACC TCC CTA 499 Pro Ala Leu Leu ProAsp Ser Pro Met Glu Gln Leu His Thr Ser Leu         95                 100                 105 CTA GGA CTC AGC CAACTC CTC CAG CCA GAG GAT CAC CCC CGG GAG ACC 547 Leu Gly Leu Ser Gln LeuLeu Gln Pro Glu Asp His Pro Arg Glu Thr    110                 115                 120 CAA CAG ATG CCC AGC CTGAGT TCT AGT CAG CAG TGG CAG CGC CCC CTT 595 Gln Gln Met Pro Ser Leu SerSer Ser Gln Gln Trp Gln Arg Pro Leu125                 130                 135                 140 CTC CGTTCC AAG ATC CTT CGA AGC CTC CAG GCC TTT TTG GCC ATA GCT 643 Leu Arg SerLys Ile Leu Arg Ser Leu Gln Ala Phe Leu Ala Ile Ala                145                 150                 155 GCC CGG GTCTTT GCC CAC GGA GCA GCA ACT CTG ACT GAG CCC TTA GTG 691 Ala Arg Val PheAla His Gly Ala Ala Thr Leu Thr Glu Pro Leu Val            160                 165                 170 CCA ACA GCTTAAGGATGCC CAGGTTCCCA TGGCTACCAT GATAAGACTA 740 Pro Thr Ala         175ATCTATCAGC CCAGACATCT ACCAGTTAAT TAACCCATTA GGACTTGTGC TGTTCTTGTT 800TCGTTTGTTT TGCGTGAAGG GCAAGGACAC CATTATTAAA GAGAAAAGAA ACAAACCCCA 860GAGCAGGCAG CTGGCTAGAG AAAGGAGCTG GAGAAGAAGA ATAAAGTCTC GAGCCCTTGG 920CCTTGGAAGC GGGCAAGCAG CTGCGTGGCC TGAGGGGAAG GGGGCGGTGG CATCGAGAAA 980CTGTGAGAAA ACCCAGAGCA TCAGAAAAAG TGAGCCCAGG CTTTGGCCAT TATCTGTAAG 1040AAAAACAAGA AAAGGGGAAC ATTATACTTT CCTGGGTGGC TCAGGGAAAT GTGCAGATGC 1100ACAGTACTCC AGACAGCAGC TCTGTACCTG CCTGCTCTGT CCCTCAGTTC TAACAGAATC 1160TAGTCACTAA GAACTAACAG GACTACCAAT ACGAACTGAC AAA 1203MLDCRAVIMLWLLPWVTQGLAVPRSSSPDWAQCQQLSRNLCMLAWNAHAPAGHMNLLREEEDEETKNNVPRIQCEDGCDPQGLKDNSQFCLQRIRQGLAFYKHLLDSDIFKGEPALLPDSPMEQLHTSLLGLSQLLQPEDHPRETQQMPSLSSSQQWQRPLLRSKILRSLQAFLAIAARVFAHGAATLTEPLVPTA

TABLE 2 Comparison of various IL6 and G-CSF embodiments compared toIL-B30. Human IL-B30 is SEQ ID NO: 2; mouse ILB30 is SEQ ID NO: 4; pigIL-B30 is SEQ ID NO: 5; bovine G-CSF is SEQ ID NO: 6; feline G-CSF isSEQ ID NO: 7; human G-CSF is SEQ ID NO: 8; mouse G-CSF is SEQ ID NO: 9;otter IL-6 is SEQ ID NO: 10; feline IL-6 is SEQ ID NO: 11; human IL-6 isSEQ ID NO: 12; sheep IL-6 is SEQ ID NO: 13; mouse IL-6 is SEQ ID NO: 14;chicken MGF is SEQ ID NO: 15; and KSHV, kaposis sarcoma herpes virus, aviral IL-6, is SEQ ID NO: 16. il30_human .......... ......VPGGSSPVWTQCQQ LSQKLCT.LA WSAHPLVG.. il30_mouse .......... ......VPRSSSPDWAQCQQ LSRNLCM.LA WNAHAPAG.. il30_pig .......... .................... .......... .......... gcsf_bovin ......TPLG P.......ARSLPQSFLLKC LEQVRKIQAD GAELQERL.. gcsf_felca ......TPLG P.......TSSLPQSFLLKC LEQVRKVQAD GTALQERL.. gcsf_human ......TPLG P.......ASSLPQSFLLKC LEQVRKIQGD GAALQEKLVS gcsf_mouse VPLVTVSALP P.......SLPLPRSFLLKS LEQVRKIQAS GSVLLEQL.. il6_otter .AFPTPGPLG GDSKDDATSNRPPLTSADKM EDFIKFILGK ISALRNEM.. il6_felca .AFPTPGPLG G....DATSNRLPLTPADKM EELIKYILGK ISALKKEM.. il6_human .AFPAPVPPG EDSKDVAAPHRQPLTSSERI DKQIRYILDG ISALRKET.. il6_sheep .AFPTPGPLG EDFKNDTTPSRLLLTTPEKT EALIKHIVDK ISAIRKEI.. il6_mouse .AFPTSQVRR GDFTEDTTPNR.PVYTTSQV GGLITHVLWE IVEMRKEL.. mgf_chick .......... .APLAELSGDHDFQLFLHKN LEFTRKIRGD VAALQRAV.. il6_khsv .......... .......TRGKLPDAPEFEK DLLIQRLNWM LWVIDECFRD il30_human .HMD.LREEG DEETTNDVPHI...QCGDGC DPQGLRDNSQ FCLQRIHQGL il30_mouse .HMNLLREEE DEETKNNVPRI...QCEDGC DPQGLKDNSQ FCLQRIRQGL il30_pig .......... .................... .......... SCLQRIHQGL gcsf_bovin .CAA.HKLCH PEELMLLRHSLGIP.QAPLS SCSSQSLQLR GCLNQLHGGL gcsf_felca .CAA.HKLCH PEELVLLGHALGIP.QAPLS SCSSQALQLT GCLRQLHSGL gcsf_human ECAT.YKLCH PEELVLLGHSLGIP.WAPLS SCPSQALQLA GCLSQLHSGL gcsf_mouse .CAT.YKLCH PEELVLLGHSLGIP.KASLS GCSSQALQQT QCLSQLHSGL il6_otter .CDK.YNKCE DSKEVLAENNLNLPKLAEKD RCFQSRFNQE TCLTRITTGL il6_felca .CDN.YNKCE DSKEALAENNLNLPKLAEKD GCFQSGFNQE TCLTRITTGL il6_human .CNK.SNMCE SSKEALAENNLNLPKMAEKD GCFQSGFNEE TCLVKIITGL il6 sheep .CEK.NDECE NSKETLAENKLKLPKMEEKD GCFQSGFNQA ICLIKTTAGL il6_mouse .CNG.NSDCM NNDDALAENNLKLPEIQRND GCYQTGYNQE ICLLKISSGL mgf_chick .CDT.FQLCT EEELQLVQPDPHLV.QAPLD QCHKRGFQAE VCFTQIRAGL il6_khsv LCYR.TGICK GILEPAAIFHLKLPAINDTD HCGLIGFNET SCLKKLADGF il30_human IFYEKLLGSD IFTGE......PSLLPDSPV AQLHASLLGL SQLLQPE..G il30_mouse AFYKHLLDSD IFKGE......PALLPDSPM EQLHTSLLGL SQLLQPE..D il30_pig VFYEKLLGSD IFTGE......PSLHPDGSV GQLHASLLGL RQLLQPE..G gcsf_bovin FLYQGLLQAL AGIS.......PELAPTLDT LQLDVTDFAT NIWLQMEDLG gcsf_felca FLYQGLLQAL AGIS.......PELAPTLDM LQLDITDFAI NIWQQMEDVG gcsf_human FLYQGLLQAL EGIS.......PELGPTLDT LQLDVADFAT TIWQQMEELG gcsf_mouse CLYQGLLQAL SGIS.......PALAPTLDL LQLDVANFAT TIWQQMENLG il6_otter QEFQIHLKYL ESNYEG...NKDNAHSVYIS TKHLLQTLRP M..NQIEVTT i16_felca QEFQIYLKFL QDKYEG...DKENAKSVYTS TNVLLQMLKR KGKNQDEVTI i16_human LEFEVYLEYL QNRFES...SEEQARAVQMS TKVLIQFLQK KAKNLDAITT il6_sheep LEYQIYLDFL QNEFEG...NQETVMELQSS IRTLIQILKE .........I i16_mouse LEYHSYLEYM KNNLKDN..KKDKARVLQRD TETLIHIFNQ EVKDLHKIVL mgf_chick HAYHDSLGAV LRLLP.......NHTTLVET LQLDAANLSS NIQQQMEDLG i16_khsv FEFEVLFKFL TTEFGKSVINVDVMELLTKT LGWDIQEELN KLTKTHY..S il30_human HHWETQQIP. .SLSPSQ..PWQRLLLRFKI LRSLQAFVAV AARVFAHGAA il30_mouse HPRETQQMP. .SLSSSQ..QWQRPLLRSKI LRSLQAFLAI AARVFAHGAA 1130_pig HHWETEQTP. .SPSPSQ..PWQRLLLRLKI LRSLQAFVAV AARVFAHGAA gcsf_bovin AAPAVQPTQ. .GAMPTFTSAFQRRAGGVLV ASQLHRFLEL AYRGLRYLAE gcsf_felca MAPAVPPTQ. .GTMPTFTSAFQRRAGGTLV ASNLQSFLEV AYRALRHFTK gcsf_human MAPALQPTQ. .GAMPAFASAFQRRAGGVLV ASHLQSFLEV SYRVLRHLAQ gcsf_mouse VAPTVQPTQ. .SAMPAFTSAFQRRAGGVLA ISYLQGFLET ARLALHHLA. il6_otter PDPTTDASL. .QALFKSQDKWLKHTTIHLI LRRLEDFLQF SLRAIRIM.. i16_felca PVPTVEVGL. .QLSCSHR.RVAEAHNNHLT LRRLEDFLQL RLRAVRIM.. i16_human PDPTTNASL. .LTKLQAQNQWLQDMTTHLI LRSFKEFLQS SLRALRQM.. il6_sheep TTPATHTDM. .LEKMQSSNEWVKNAKVIII LRSLENFLQF SLRAIRNK.. il6_mouse PTPISNALL. .TDKLESQKEWLRTKTIQFI LKSLEEFLKV TLRSTRQT.. mgf_chick LDTVTLPAEQ RSPPPTFSGPFQQQVGGFFI LANFQRFLET AYRALRHLAR il6_khsv P.PKFDRG.. LLGRLQGLKYWVRHFASFYV LSAMEKFAGQ AVRVLDSIPD il30_human TLSP...... il30_mouseTLTEPLVPTA il30_pig TLSQ...... gcsf_bovin P....... gcsf_felca P.......gcsf_human P....... gcsf_mouse ........ il6_otter ........ il6_felca........ il6_human ........ il6_sheep ........ i16_mouse ........mgf_chick L....... il6_khsv VTPDVHDK

The structural homology of IL-B30 to related cytokine proteins suggestsrelated function of this molecule. IL-B30 is a long chain cytokineexhibiting sequence similarity to IL-6 and G-CSF.

IL-B30 agonists, or antagonists, may also act as functional or receptorantagonists, e.g., which block IL-6 or G-CSF binding to their respectivereceptors, or mediating the opposite actions. Thus, IL-B30, or itsantagonists, may be useful in the treatment of abnormal medicalconditions, including immune disorders, e.g., T cell immunedeficiencies, chronic inflammation, or tissue rejection, or incardiovascular or neurophysiological conditions.

The natural antigens are capable of mediating various biochemicalresponses which lead to biological or physiological responses in targetcells. The preferred embodiment characterized herein is from human, butother primate, or other species counterparts exist in nature. Additionalsequences for proteins in other mammalian species, e.g., primates,canines, felines, and rodents, should also be available. See below. Thedescriptions below are directed, for exemplary purposes, to a humanIL-B30, but are likewise applicable to related embodiments from otherspecies.

II. Purified IL-B30

Human IL-B30 amino acid sequence, is shown as one embodiment within SEQID NO: 2. Other naturally occurring nucleic acids which encode theprotein can be isolated by standard procedures using the providedsequence, e.g., PCR techniques, or by hybridization. These amino acidsequences, provided amino to carboxy, are important in providingsequence information for the cytokine allowing for distinguishing theprotein antigen from other proteins and exemplifying numerous variants.Moreover, the peptide sequences allow preparation of peptides togenerate antibodies to recognize such segments, and nucleotide sequencesallow preparation of oligonucleotide probes, both of which arestrategies for detection or isolation, e.g., cloning, of genes encodingsuch sequences.

As used herein, the term “human soluble IL-B30” shall encompass, whenused in a protein context, a protein having amino acid sequencecorresponding to a soluble polypeptide shown in SEQ ID NO: 2, orsignificant fragments thereof. Preferred embodiments comprise aplurality of distinct, e.g., nonoverlapping, segments of the specifiedlength. Typically, the plurality will be at least two, more usually atleast three, and preferably 5, 7, or even more. While the length minimaare provided, longer lengths, of various sizes, may be appropriate,e.g., one of length 7, and two of length 12.

Binding components, e.g., antibodies, typically bind to an IL-B30 withhigh affinity, e.g., at least about 100 nM, usually better than about 30nM, preferably better than about 10 nM, and more preferably at betterthan about 3 nM. Counterpart proteins will be found in mammalian speciesother than human, e.g., other primates, ungulates, or rodents.Non-mammalian species should also possess structurally or functionallyrelated genes and proteins, e.g., birds or amphibians.

The term “polypeptide” as used herein includes a significant fragment orsegment, and encompasses a stretch of amino acid residues of at leastabout 8 amino acids, generally at least about 12 amino acids, typicallyat least about 16 amino acids, preferably at least about 20 amino acids,and, in particularly preferred embodiments, at least about 30 or moreamino acids, e.g., 35, 40, 45, 50, etc. Such fragments may have endswhich begin and/or end at virtually all positions, e.g., beginning atresidues 1, 2, 3, etc., and ending at, e.g., 150, 149, 148, etc., in allpractical combinations. Particularly interesting peptides have endscorresponding to structural domain boundaries, e.g., helices A, B, C,and/or D. See Table 1.

The term “binding composition” refers to molecules that bind withspecificity to IL-B30, e.g., in an antibody-antigen interaction. Thespecificity may be more or less inclusive, e.g., specific to aparticular embodiment, or to groups of related embodiments, e.g.,primate, rodent, etc. It also includes compounds, e.g., proteins, whichspecifically associate with IL-B30, including in a naturalphysiologically relevant protein-protein interaction, either covalent ornon-covalent. The molecule may be a polymer, or chemical reagent. Afunctional analog may be a protein with structural modifications, or itmay be a molecule which has a molecular shape which interacts with theappropriate binding determinants. The compounds may serve as agonists orantagonists of a receptor binding interaction, see, e.g., Goodman, etal. (eds.) Goodman & Gilman's: The Pharmacological Bases of Therapeutics(current ed.) Pergamon Press.

Substantially pure, e.g., in a protein context, typically means that theprotein is free from other contaminating proteins, nucleic acids, orother biologicals derived from the original source organism. Purity maybe assayed by standard methods, typically by weight, and will ordinarilybe at least about 40% pure, generally at least about 50% pure, often atleast about 60% pure, typically at least about 80% pure, preferably atleast about 90% pure, and in most preferred embodiments, at least about95% pure. Carriers or excipients will often be added.

Solubility of a polypeptide or fragment depends upon the environment andthe polypeptide. Many parameters affect polypeptide solubility,including temperature, electrolyte environment, size and molecularcharacteristics of the polypeptide, and nature of the solvent.Typically, the temperature at which the polypeptide is used ranges fromabout 4° C. to about 65° C. Usually the temperature at use is greaterthan about 18° C. For diagnostic purposes, the temperature will usuallybe about room temperature or warmer, but less than the denaturationtemperature of components in the assay. For therapeutic purposes, thetemperature will usually be body temperature, typically about 37° C. forhumans and mice, though under certain situations the temperature may beraised or lowered in situ or in vitro.

The size and structure of the polypeptide should generally be in asubstantially stable state, and usually not in a denatured state. Thepolypeptide may be associated with other polypeptides in a quaternarystructure, e.g., to confer solubility, or associated with lipids ordetergents.

The solvent and electrolytes will usually be a biologically compatiblebuffer, of a type used for preservation of biological activities, andwill usually approximate a physiological aqueous solvent. Usually thesolvent will have a neutral pH, typically between about 5 and 10, andpreferably about 7.5. On some occasions, one or more detergents will beadded, typically a mild non-denaturing one, e.g., CHS (cholesterylhemisuccinate) or CHAPS (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), or a low enough concentration as to avoid significantdisruption of structural or physiological properties of the protein. Inother instances, a harsh detergent may be used to effect significantdenaturation.

III. Physical Variants

This invention also encompasses proteins or peptides having substantialamino acid sequence identity with the amino acid sequence of the IL-B30antigen. The variants include species, polymorphic, or allelic variants.

Amino acid sequence homology, or sequence identity, is determined byoptimizing residue matches, if necessary, by introducing gaps asrequired. See also Needleham, et al. (1970) J. Mol. Biol. 48:443-453;Sankoff, et al. (1983) Chapter One in Time Warps, String Edits, andMacromolecules: The Theory and Practice of Sequence Comparison,Addison-Wesley, Reading, Mass.; and software packages fromIntelliGenetics, Mountain View, Calif.; and the University of WisconsinGenetics Computer Group, Madison, Wis. Sequence identity changes whenconsidering conservative substitutions as matches. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine; valine, isoleucine, leucine; aspartic acid,glutamic acid; asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine. The conservation may apply tobiological features, functional features, or structural features.Homologous amino acid sequences are typically intended to includenatural polymorphic or allelic and interspecies variations of a proteinsequence. Typical homologous proteins or peptides will have from 25-100%identity (if gaps can be introduced), to 50-100% identity (ifconservative substitutions are included) with the amino acid sequence ofthe IL-B30. Identity measures will be at least about 35%, generally atleast about 40%, often at least about 50%, typically at least about 60%,usually at least about 70%, preferably at least about 80%, and morepreferably at least about 90%.

The isolated IL-B30 DNA can be readily modified by nucleotidesubstitutions, nucleotide deletions, nucleotide insertions, andinversions of short nucleotide stretches. These modifications result innovel DNA sequences which encode these antigens, their derivatives, orproteins having similar physiological, immunogenic, antigenic, or otherfunctional activity. These modified sequences can be used to producemutant antigens or to enhance expression. Enhanced expression mayinvolve gene amplification, increased transcription, increasedtranslation, and other mechanisms. “Mutant IL-B30” encompasses apolypeptide otherwise falling within the sequence identity definition ofthe IL-B30 as set forth above, but having an amino acid sequence whichdiffers from that of IL-B30 as normally found in nature, whether by wayof deletion, substitution, or insertion. This generally includesproteins having significant identity with a protein having sequence ofSEQ ID NO: 2, and as sharing various biological activities, e.g.,antigenic or immunogenic, with those sequences, and in preferredembodiments contain most of the natural full length disclosed sequences.Full length sequences will typically be preferred, though truncatedversions will also be useful, likewise, genes or proteins found fromnatural sources are typically most desired. Similar concepts apply todifferent IL-B30 proteins, particularly those found in various warmblooded animals, e.g., mammals and birds. These descriptions aregenerally meant to encompass all IL-B30 proteins, not limited to theparticular primate embodiments specifically discussed.

IL-B30 mutagenesis can also be conducted by making amino acid insertionsor deletions. Substitutions, deletions, insertions, or any combinationsmay be generated to arrive at a final construct. Insertions includeamino- or carboxy-terminal fusions. Random mutagenesis can be conductedat a target codon and the expressed mutants can then be screened for thedesired activity. Methods for making substitution mutations atpredetermined sites in DNA having a known sequence are well known in theart, e.g., by M13 primer mutagenesis or polymerase chain reaction (PCR)techniques. See, e.g., Sambrook, et al. (1989); Ausubel, et al. (1987and Supplements); and Kunkel, et al. (1987) Methods in Enzymol.154:367-382. Preferred embodiments include, e.g., 1-fold, 2-fold,3-fold, 5-fold, 7-fold, etc., preferably conservative substitutions atthe nucleotide or amino acid levels. Preferably the substitutions willbe away from the conserved cysteines, and often will be in the regionsaway from the helical structural domains. Such variants may be useful toproduce specific antibodies, and often will share many or all biologicalproperties.

The present invention also provides recombinant proteins, e.g.,heterologous fusion proteins using segments from these proteins. Aheterologous fusion protein is a fusion of proteins or segments whichare naturally not normally fused in the same manner. A similar conceptapplies to heterologous nucleic acid sequences.

In addition, new constructs may be made from combining similarfunctional domains from other proteins. For example, target-binding orother segments may be “swapped” between different new fusionpolypeptides or fragments. See, e.g., Cunningham, et al. (1989) Science243:1330-1336; and O'Dowd, et al. (1988) J. Biol. Chem. 263:15985-15992.

The phosphoramidite method described by Beaucage and Carruthers (1981)Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNAfragments. A double stranded fragment will often be obtained either bysynthesizing the complementary strand and annealing the strand togetherunder appropriate conditions or by adding the complementary strand usingDNA polymerase with an appropriate primer sequence, e.g., PCRtechniques.

Structural analysis can be applied to this gene, in comparison to theIL-6 family of cytokines. The family includes, e.g., IL-6, IL-11, IL-12,G-CSF, LIF, OSM, CNTF, and Ob. Alignment of the human, pig, and mouseIL-B30 sequences with other members of the IL-6 family should allowdefinition of structural features. In particular, β-sheet and α-helixresidues can be determined using, e.g., RASMOL program, see Bazan, etal. (1996) Nature 379:591; Lodi, et al. (1994) Science 263:1762-1766;Sayle and Milner-White (1995) TIBS 20:374-376; and Gronenberg, et al.(1991) Protein Engineering 4:263-269. Preferred residues forsubstitutions include the surface exposed residues which would bepredicted to interact with receptor. Other residues which shouldconserve function will be conservative substitutions, particularly atposition far from the surface exposed residues.

IV. Functional Variants

The blocking of physiological response to IL-B30s may result from thecompetitive inhibition of binding of the ligand to its receptor.

In vitro assays of the present invention will often use isolatedprotein, soluble fragments comprising receptor binding segments of theseproteins, or fragments attached to solid phase substrates. These assayswill also allow for the diagnostic determination of the effects ofeither binding segment mutations and modifications, or cytokinemutations and modifications, e.g., IL-B30 analogs.

This invention also contemplates the use of competitive drug screeningassays, e.g., where neutralizing antibodies to the cytokine, or receptorbinding fragments compete with a test compound.

“Derivatives” of IL-B30 antigens include amino acid sequence mutantsfrom naturally occurring forms, glycosylation variants, and covalent oraggregate conjugates with other chemical moieties. Covalent derivativescan be prepared by linkage of functionalities to groups which are foundin IL-B30 amino acid side chains or at the N- or C-termini, e.g., bystandard means. See, e.g., Lundblad and Noyes (1988) Chemical Reagentsfor Protein Modification, vols. 1-2, CRC Press, Inc., Boca Raton, Fla.;Hugli (ed. 1989) Techniues in Protein Chemistry, Academic Press, SanDiego, Calif.; and Wong (1991) Chemistry of Protein Conjugation andCross Linking, CRC Press, Boca Raton, Fla.

In particular, glycosylation alterations are included, e.g., made bymodifying the glycosylation patterns of a polypeptide during itssynthesis and processing, or in further processing steps. See, e.g.,Elbein (1987) Ann. Rev. Biochem. 56:497-534. Also embraced are versionsof the peptides with the same primary amino acid sequence which haveother minor modifications, including phosphorylated amino acid residues,e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

Fusion polypeptides between IL-B30s and other homologous or heterologousproteins are also provided. Many cytokine receptors or other surfaceproteins are multimeric, e.g., homodimeric entities, and a repeatconstruct may have various advantages, including lessened susceptibilityto proteolytic cleavage. Typical examples are fusions of a reporterpolypeptide, e.g., luciferase, with a segment or domain of a protein,e.g., a receptor-binding segment, so that the presence or location ofthe fused ligand may be easily determined. See, e.g., Dull, et al., U.S.Pat. No. 4,859,609. Other gene fusion partners include bacterialβ-galactosidase, trpE, Protein A, β-lactamase, alpha amylase, alcoholdehydrogenase, yeast alpha mating factor, and detection or purificationtags such as a FLAG sequence of His6 sequence. See, e.g., Godowski, etal. (1988) Science 241:812-816.

Fusion peptides will typically be made by either recombinant nucleicacid methods or by synthetic polypeptide methods. Techniques for nucleicacid manipulation and expression are described generally, e.g., inSambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.),vols. 1-3, Cold Spring Harbor Laboratory; and Ausubel, et al. (eds.1993) Current Protocols in Molecular Biology, Greene and Wiley, NY.Techniques for synthesis of polypeptides are described, e.g., inMerrifield (1963) J. Amer. Chem. Soc. 85:2149-2156; Merrifield (1986)Science 232: 341-347; Atherton, et al. (1989) Solid Phase PeptideSynthesis: A Practical Approach, IRL Press, Oxford; and Grant (1992)Synthetic Peptides: A User's Guide, W.H. Freeman, NY. Refolding methodsmay be applicable to synthetic proteins.

This invention also contemplates the use of derivatives of IL-B30proteins other than variations in amino acid sequence or glycosylation.Such derivatives may involve covalent or aggregative association withchemical moieties or protein carriers. Covalent or aggregativederivatives will be useful as immunogens, as reagents in immunoassays,or in purification methods such as for affinity purification of bindingpartners, e.g., other antigens. An IL-B30 can be immobilized by covalentbonding to a solid support such as cyanogen bromide-activated SEPHAROSE,by methods which are well known in the art, or adsorbed onto polyolefinsurfaces, with or without glutaraldehyde cross-linking, for use in theassay or purification of anti-IL-B30 antibodies or an alternativebinding composition. The IL-B30 proteins can also be labeled with adetectable group, e.g., for use in diagnostic assays. Purification ofIL-B30 may be effected by an immobilized antibody or complementarybinding partner, e.g., binding portion of a receptor.

A solubilized IL-B30 or fragment of this invention can be used as animmunogen for the production of antisera or antibodies specific forbinding. Purified antigen can be used to screen monoclonal antibodies orantigen-binding fragments, encompassing antigen binding fragments ofnatural antibodies, e.g., Fab, Fab′, F(ab)₂, etc. Purified IL-B30antigens can also be used as a reagent to detect antibodies generated inresponse to the presence of elevated levels of the cytokine, which maybe diagnostic of an abnormal or specific physiological or diseasecondition. This invention contemplates antibodies raised against aminoacid sequences encoded by nucleotide sequence shown in SEQ ID NO: 1, orfragments of proteins containing it. In particular, this inventioncontemplates antibodies having binding affinity to or being raisedagainst specific domains, e.g., helices A, B, C, or D.

The present invention contemplates the isolation of additional closelyrelated species variants. Southern and Northern blot analysis willestablish that similar genetic entities exist in other mammals. It islikely that IL-B30s are widespread in species variants, e.g., rodents,lagomorphs, carnivores, artiodactyla, perissodactyla, and primates.

The invention also provides means to isolate a group of related antigensdisplaying both distinctness and similarities in structure, expression,and function. Elucidation of many of the physiological effects of themolecules will be greatly accelerated by the isolation andcharacterization of additional distinct species or polymorphic variantsof them. In particular, the present invention provides useful probes foridentifying additional homologous genetic entities in different species.

The isolated genes will allow transformation of cells lacking expressionof an IL-B30, e.g., either species types or cells which lackcorresponding proteins and exhibit negative background activity. Thisshould allow analysis of the function of IL-B30 in comparison tountransformed control cells.

Dissection of critical structural elements which effect the variousphysiological functions mediated through these antigens is possibleusing standard techniques of modern molecular biology, particularly incomparing members of the related class. See, e.g., the homolog-scanningmutagenesis technique described in Cunningham, et al. (1989) Science243:1339-1336; and approaches used in O'Dowd, et al. (1988) J. Biol.Chem. 263:15985-15992; and Lechleiter, et al. (1990) EMBO J.9:4381-4390.

Intracellular functions would probably involve receptor signaling.However, protein internalization may occur under certain circumstances,and interaction between intracellular components and cytokine may occur.Specific segments of interaction of IL-B30 with interacting componentsmay be identified by mutagenesis or direct biochemical means, e.g.,cross-linking or affinity methods. Structural analysis bycrystallographic or other physical methods will also be applicable.Further investigation of the mechanism of signal transduction willinclude study of associated components which may be isolatable byaffinity methods or by genetic means, e.g., complementation analysis ofmutants.

Further study of the expression and control of IL-B30 will be pursued.The controlling elements associated with the antigens should exhibitdifferential physiological, developmental, tissue specific, or otherexpression patterns. Upstream or downstream genetic regions, e.g.,control elements, are of interest.

Structural studies of the IL-B30 antigens will lead to design of newantigens, particularly analogs exhibiting agonist or antagonistproperties on the molecule. This can be combined with previouslydescribed screening methods to isolate antigens exhibiting desiredspectra of activities.

V. Antibodies

Antibodies can be raised to various epitopes of the IL-B30 proteins,including species, polymorphic, or allelic variants, and fragmentsthereof, both in their naturally occurring forms and in theirrecombinant forms. Additionally, antibodies can be raised to IL-B30s ineither their active forms or in their inactive forms, including nativeor denatured versions. Anti-idiotypic antibodies are also contemplated.

Antibodies, including binding fragments and single chain versions,against predetermined fragments of the antigens can be raised byimmunization of animals with conjugates of the fragments withimmunogenic proteins. Monoclonal antibodies are prepared from cellssecreting the desired antibody. These antibodies can be screened forbinding to normal or defective IL-B30s, or screened for agonistic orantagonistic activity, e.g., mediated through a receptor. Antibodies maybe agonistic or antagonistic, e.g., by sterically blocking binding to areceptor. These monoclonal antibodies will usually bind with at least aK_(D) of about 1 mM, more usually at least about 300 μM, typically atleast about 100 μM, more typically at least about 30 μM, preferably atleast about 10 μM, and more preferably at least about 3 μM or better.

The antibodies of this invention can also be useful in diagnosticapplications. As capture or non-neutralizing antibodies, they can bescreened for ability to bind to the antigens without inhibiting bindingto a receptor. As neutralizing antibodies, they can be useful incompetitive binding assays. They will also be useful in detecting orquantifying IL-B30 protein or its receptors. See, e.g., Chan (ed. 1987)Immunology: A Practical Guide, Academic Press, Orlando, Fla.; Price andNewman (eds. 1991) Principles and Practice of Immunoassay, StocktonPress, N.Y.; and Ngo (ed. 1988) Nonisotopic Immunoassay, Plenum Press,N.Y. Cross absorptions or other tests will identify antibodies whichexhibit various spectra of specificities, e.g., unique or shared speciesspecificities.

Further, the antibodies, including antigen binding fragments, of thisinvention can be potent antagonists that bind to the antigen and inhibitfunctional binding, e.g., to a receptor which may elicit a biologicalresponse. They also can be useful as non-neutralizing antibodies and canbe coupled to toxins or radionuclides so that when the antibody binds toantigen, a cell expressing it, e.g., on its surface, is killed. Further,these antibodies can be conjugated to drugs or other therapeutic agents,either directly or indirectly by means of a linker, and may effect drugtargeting.

Antigen fragments may be joined to other materials, particularlypolypeptides, as fused or covalently joined polypeptides to be used asimmunogens. An antigen and its fragments may be fused or covalentlylinked to a variety of immunogens, such as keyhole limpet hemocyanin,bovine serum albumin, tetanus toxoid, etc. See Microbiology, HoeberMedical Division, Harper and Row, 1969; Landsteiner (1962) Specificityof Serological Reactions, Dover Publications, New York; Williams, et al.(1967) Methods in Immunology and Immunochemistry, vol. 1, AcademicPress, New York; and Harlow and Lane (1988) Antibodies: A LaboratoryManual, CSH Press, NY, for descriptions of methods of preparingpolyclonal antisera.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious mammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies maybe found in, e.g., Stites, et al. (eds.) Basic and Clinical Immunology(4th ed.), Lange Medical Publications, Los Altos, Calif., and referencescited therein; Harlow and Lane (1988) Antibodies: A Laboratory Manual,CSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice(2d ed.), Academic Press, New York; and particularly in Kohler andMilstein (1975) in Nature 256:495-497, which discusses one method ofgenerating monoclonal antibodies.

Other suitable techniques involve in vitro exposure of lymphocytes tothe antigenic polypeptides or alternatively to selection of libraries ofantibodies in phage or similar vectors. See, Huse, et al. (1989)“Generation of a Large Combinatorial Library of the ImmunoglobulinRepertoire in Phage Lambda,” Science 246:1275-1281; and Ward, et al.(1989) Nature 341:544-546. The polypeptides and antibodies of thepresent invention may be used with or without modification, includingchimeric or humanized antibodies. Frequently, the polypeptides andantibodies will be labeled by joining, either covalently ornon-covalently, a substance which provides for a detectable signal. Awide variety of labels and conjugation techniques are known and arereported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Patents, teaching the use of such labelsinclude U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulinsmay be produced, see Cabilly, U.S. Pat. No. 4,816,567; Moore, et al.,U.S. Pat. No. 4,642,334; and Queen, et al. (1989) Proc. Nat'l Acad. Sci.USA 86:10029-10033.

The antibodies of this invention can also be used for affinitychromatography in isolating the protein. Columns can be prepared wherethe antibodies are linked to a solid support. See, e.g., Wilchek et al.(1984) Meth. Enzymol. 104:3-55.

Antibodies raised against each IL-B30 will also be useful to raiseanti-idiotypic antibodies. These will be useful in detecting ordiagnosing various immunological conditions related to expression of therespective antigens.

VI. Nucleic Acids

The described peptide sequences and the related reagents are useful indetecting, isolating, or identifying a DNA clone encoding IL-B30, e.g.,from a natural source. Typically, it will be useful in isolating a genefrom mammal, and similar procedures will be applied to isolate genesfrom other species, e.g., warm blooded animals, such as birds andmammals. Cross hybridization will allow isolation of IL-B30 from thesame, e.g., polymorphic variants, or other species. A number ofdifferent approaches will be available to successfully isolate asuitable nucleic acid clone.

The purified protein or defined peptides are useful for generatingantibodies by standard methods, as described above. Synthetic peptidesor purified protein can be presented to an immune system to generatemonoclonal or polyclonal antibodies. See, e.g., Coligan (1991) CurrentProtocols in Immunology Wiley/Greene; and Harlow and Lane (1989)Antibodies: A Laboratory Manual, Cold Spring Harbor Press.

For example, the specific binding composition could be used forscreening of an expression library made from a cell line which expressesan IL-B30. Screening of intracellular expression can be performed byvarious staining or immunofluorescence procedures. Binding compositionscould be used to affinity purify or sort out cells expressing a surfacefusion protein.

The peptide segments can also be used to predict appropriateoligonucleotides to screen a library. The genetic code can be used toselect appropriate oligonucleotides useful as probes for screening. See,e.g., SEQ ID NO: 1. In combination with polymerase chain reaction (PCR)techniques, synthetic oligonucleotides will be useful in selectingcorrect clones from a library.

Complementary sequences will also be used as probes, primers, orantisense strands. Various fragments should be particularly useful,e.g., coupled with anchored vector or poly-A complementary PCRtechniques or with complementary DNA of other peptides.

This invention contemplates use of isolated DNA or fragments to encode abiologically active corresponding IL-B30 polypeptide, particularlylacking the portion coding the untranslated 5′ portion of the describedsequence. In addition, this invention covers isolated or recombinant DNAwhich encodes a biologically active protein or polypeptide and which iscapable of hybridizing under appropriate conditions with the DNAsequences described herein. Said biologically active protein orpolypeptide can be an intact antigen, or fragment, and have an aminoacid sequence disclosed in, e.g., SEQ ID NO: 2, particularly a mature,secreted polypeptide. Further, this invention covers the use of isolatedor recombinant DNA, or fragments thereof, which encode proteins whichexhibit high identity to a secreted IL-B30. The isolated DNA can havethe respective regulatory sequences in the 5′ and 3′ flanks, e.g.,promoters, enhancers, poly-A addition signals, and others.Alternatively, expression may be effected by operably linking a codingsegment to a heterologous promoter, e.g., by inserting a promoterupstream from an endogenous gene.

An “isolated” nucleic acid is a nucleic acid, e.g., an RNA, DNA, or amixed polymer, which is substantially separated from other componentswhich naturally accompany a native sequence, e.g., ribosomes,polymerases, and/or flanking genomic sequences from the originatingspecies. The term embraces a nucleic acid sequence which has beenremoved from its naturally occurring environment, and includesrecombinant or cloned DNA isolates and chemically synthesized analogs oranalogs biologically synthesized by heterologous systems. Asubstantially pure molecule includes isolated forms of the molecule.Generally, the nucleic acid will be in a vector or fragment less thanabout 50 kb, usually less than about 30 kb, typically less than about 10kb, and preferably less than about 6 kb.

An isolated nucleic acid will generally be a homogeneous composition ofmolecules, but will, in some embodiments, contain minor heterogeneity.This heterogeneity is typically found at the polymer ends or portionsnot critical to a desired biological function or activity.

A “recombinant” nucleic acid is defined either by its method ofproduction or its structure. In reference to its method of production,e.g., a product made by a process, the process is use of recombinantnucleic acid techniques, e.g., involving human intervention in thenucleotide sequence, typically selection or production. Alternatively,it can be a nucleic acid made by generating a sequence comprising fusionof two fragments which are not naturally contiguous to each other, butis meant to exclude products of nature, e.g., naturally occurringmutants. Thus, e.g., products made by transforming cells with anyunnaturally occurring vector is encompassed, as are nucleic acidscomprising sequence derived using any synthetic oligonucleotide process.Such is often done to replace a codon with a redundant codon encodingthe same or a conservative amino acid, while typically introducing orremoving a sequence recognition site.

Alternatively, it is performed to join together nucleic acid segments ofdesired functions to generate a single genetic entity comprising adesired combination of functions not found in the commonly availablenatural forms. Restriction enzyme recognition sites are often the targetof such artificial manipulations, but other site specific targets, e.g.,promoters, DNA replication sites, regulation sequences, controlsequences, or other useful features may be incorporated by design. Asimilar concept is intended for a recombinant, e.g., fusion,polypeptide. Specifically included are synthetic nucleic acids which, bygenetic code redundancy, encode polypeptides similar to fragments ofthese antigens, and fusions of sequences from various different speciesor polymorphic variants.

A significant “fragment” in a nucleic acid context is a contiguoussegment of at least about 17 nucleotides, generally at least about 22nucleotides, ordinarily at least about 29 nucleotides, more often atleast about 35 nucleotides, typically at least about 41 nucleotides,usually at least about 47 nucleotides, preferably at least about 55nucleotides, and in particularly preferred embodiments will be at leastabout 60 or more nucleotides, e.g., 67, 73, 81, 89, 95, etc.

A DNA which codes for an IL-B30 protein will be particularly useful toidentify genes, mRNA, and cDNA species which code for related or similarproteins, as well as DNAs which code for homologous proteins fromdifferent species. There will be homologs in other species, includingprimates, rodents, canines, felines, and birds. Various IL-B30 proteinsshould be homologous and are encompassed herein. However, even proteinsthat have a more distant evolutionary relationship to the antigen canreadily be isolated under appropriate conditions using these sequencesif they are sufficiently homologous. Primate IL-B30 proteins are ofparticular interest.

Recombinant clones derived from the genomic sequences, e.g., containingintrons, will be useful for transgenic studies, including, e.g.,transgenic cells and organisms, and for gene therapy. See, e.g., Goodnow(1992) “Transgenic Animals” in Roitt (ed.) Encyclopedia of Immunology,Academic Press, San Diego, pp. 1502-1504; Travis (1992) Science256:1392-1394; Kuhn, et al. (1991) Science 254:707-710; Capecchi (1989)Science 244:1288; Robertson (1987)(ed.) Teratocarcinomas and EmbryonicStem Cells: A Practical Approach, IRL Press, Oxford; and Rosenberg(1992) J. Clinical Oncology 10:180-199.

Substantial homology, e.g., identity, in the nucleic acid sequencecomparison context means either that the segments, or theircomplementary strands, when compared, are identical when optimallyaligned, with appropriate nucleotide insertions or deletions, in atleast about 50% of the nucleotides, generally at least about 58%,ordinarily at least about 65%, often at least about 71%, typically atleast about 77%, usually at least about 85%, preferably at least about95 to 98% or more, and in particular embodiments, as high as about 99%or more of the nucleotides. Alternatively, substantial homology existswhen the segments will hybridize under selective hybridizationconditions, to a strand, or its complement, typically using a sequenceof IL-B30, e.g., in SEQ ID NO: 1. Typically, selective hybridizationwill occur when there is at least about 55% identity over a stretch ofat least about 30 nucleotides, preferably at least about 75% over astretch of about 25 nucleotides, and most preferably at least about 90%over about 20 nucleotides. See, Kanehisa (1984) Nuc. Acids Res.12:203-213. The length of identity comparison, as described, may be overlonger stretches, and in certain embodiments will be over a stretch ofat least about 17 nucleotides, usually at least about 28 nucleotides,typically at least about 40 nucleotides, and preferably at least about75 to 100 or more nucleotides.

Stringent conditions, in referring to homology in the hybridizationcontext, will be stringent combined conditions of salt, temperature,organic solvents, and other parameters, typically those controlled inhybridization reactions. Stringent temperature conditions will usuallyinclude temperatures in excess of about 30° C., usually in excess ofabout 37° C., typically in excess of about 55° C., preferably in excessof about 70° C. Stringent salt conditions will ordinarily be less thanabout 1000 mM, usually less than about 400 mM, typically less than about250 mM, preferably less than about 150 mM, including about 100, 50, oreven 20 mM. However, the combination of parameters is much moreimportant than the measure of any single parameter. See, e.g., Wetmurand Davidson (1968) J. Mol. Biol. 31:349-370. Hybridization understringent conditions should give a background of at least 2-fold overbackground, preferably at least 3-5 or more.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optical alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith and Waterman (1981) Adv. Appl.Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch(1970) J. Mol. Biol. 48:443, by the search for similarity method ofPearson and Lipman (1988) Proc. Nat'l Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by visual inspection (seegenerally Ausubel et al., supra).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendrogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng and Doolittle (1987) J. Mol. Evol.35:351-360. The method used is similar to the method described byHiggins and Sharp (1989) CABIOS 5:151-153. The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described Altschul, et al. (1990) J. Mol. Biol. 215:403-410. Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (http:www.ncbi.nlm.nih.gov/). Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence, whicheither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul, et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are thenextended in both directions along each sequence for as far as thecumulative alignment score can be increased. Extension of the word hitsin each direction are halted when: the cumulative alignment score fallsoff by the quantity X from its maximum achieved value; the cumulativescore goes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLAST program uses asdefaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Nat'l Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparisonof both strands.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin and Altschul (1993) Proc. Nat'l Acad.Sci. USA 90:5873-5787). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences of polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid, as described below. Thus, apolypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described below.

IL-B30 from other mammalian species can be cloned and isolated bycross-species hybridization of closely related species. Homology may berelatively low between distantly related species, and thus hybridizationof relatively closely related species is advisable. Alternatively,preparation of an antibody preparation which exhibits less speciesspecificity may be useful in expression cloning approaches.

VII. Making IL-B30; Mimetics

DNA which encodes the IL-B30 or fragments thereof can be obtained bychemical synthesis, screening cDNA libraries, or screening genomiclibraries prepared from a wide variety of cell lines or tissue samples.See, e.g., Okayama and Berg (1982) Mol. Cell. Biol. 2:161-170; Gublerand Hoffman (1983) Gene 25:263-269; and Glover (ed. 1984) DNA Cloning: APractical Approach, IRL Press, Oxford. Alternatively, the sequencesprovided herein provide useful PCR primers or allow synthetic or otherpreparation of suitable genes encoding an IL-B30; including naturallyoccurring embodiments.

This DNA can be expressed in a wide variety of host cells for thesynthesis of a full-length IL-B30 or fragments which can in turn, e.g.,be used to generate polyclonal or monoclonal antibodies; for bindingstudies; for construction and expression of modified molecules; and forstructure/function studies.

Vectors, as used herein, comprise plasmids, viruses, bacteriophage,integratable DNA fragments, and other vehicles which enable theintegration of DNA fragments into the genome of the host. See, e.g.,Pouwels, et al. (1985 and Supplements) Cloning Vectors: A LaboratoryManual, Elsevier, N.Y.; and Rodriguez, et al. (eds. 1988) Vectors: ASurvey of Molecular Cloning Vectors and Their Uses, Buttersworth,Boston, Mass.

For purposes of this invention, DNA sequences are operably linked whenthey are functionally related to each other. For example, DNA for apresequence or secretory leader is operably linked to a polypeptide ifit is expressed as a preprotein or participates in directing thepolypeptide to the cell membrane or in secretion of the polypeptide. Apromoter is operably linked to a coding sequence if it controls thetranscription of the polypeptide; a ribosome binding site is operablylinked to a coding sequence if it is positioned to permit translation.Usually, operably linked means contiguous and in reading frame, however,certain genetic elements such as repressor genes are not contiguouslylinked but still bind to operator sequences that in turn controlexpression. See, e.g., Rodriguez, et al., Chapter 10, pp. 205-236;Balbas and Bolivar (1990) Methods in Enzymology 185:14-37; and Ausubel,et al. (1993) Current Protocols in Molecular Biology, Greene and Wiley,NY.

Representative examples of suitable expression vectors include pcDNA1;pCD, see Okayama, et al. (1985) Mol. Cell. Biol. 5:1136-1142; pMC1neoPoly-A, see Thomas, et al. (1987) Cell 51:503-512; and a baculovirusvector such as pAC 373 or pAC 610. See, e.g., Miller (1988) Ann. Rev.Microbiol. 42:177-199.

It will often be desired to express an IL-B30 polypeptide in a systemwhich provides a specific or defined glycosylation pattern. See, e.g.,Luckow and Summers (1988) Bio/Technology 6:47-55; and Kaufman (1990)Meth. Enzymol. 185:487-511.

The IL-B30, or a fragment thereof, may be engineered to be phosphatidylinositol (PI) linked to a cell membrane, but can be removed frommembranes by treatment with a phosphatidyl inositol cleaving enzyme,e.g., phosphatidyl inositol phospholipase-C. This releases the antigenin a biologically active form, and allows purification by standardprocedures of protein chemistry. See, e.g., Low (1989) Biochim. Biophys.Acta 988:427-454; Tse, et al. (1985) Science 230:1003-1008; and Brunner,et al. (1991) J. Cell Biol. 114:1275-1283.

Now that the IL-B30 has been characterized, fragments or derivativesthereof can be prepared by conventional processes for synthesizingpeptides. These include processes such as are described in Stewart andYoung (1984) Solid Phase Peptide Synthesis, Pierce Chemical Co.,Rockford, Ill.; Bodanszky and Bodanszky (1984) The Practice of PeptideSynthesis, Springer-Verlag, New York; Bodanszky (1984) The Principles ofPeptide Synthesis, Springer-Verlag, New York; and Villafranca (ed. 1991)Techniues in Protein Chemistry II, Academic Press, San Diego, Calif.

VIII. Uses

The present invention provides reagents which will find use indiagnostic applications as described elsewhere herein, e.g., in IL-B30mediated conditions, or below in the description of kits for diagnosis.The gene may be useful in forensic sciences, e.g., to distinguish rodentfrom human, or as a marker to distinguish between different cellsexhibiting differential expression or modification patterns.

This invention also provides reagents with significant commercial and/ortherapeutic potential. The IL-B30 (naturally occurring or recombinant),fragments thereof, and antibodies thereto, along with compoundsidentified as having binding affinity to IL-B30, should be useful asreagents for teaching techniques of molecular biology, immunology, orphysiology. Appropriate kits may be prepared with the reagents, e.g., inpractical laboratory exercises in production or use of proteins,antibodies, cloning methods, histology, etc.

The reagents will also be useful in the treatment of conditionsassociated with abnormal physiology or development, includinginflammatory conditions. They may be useful in vitro tests for presenceor absence of interacting components, which may correlate with successof particular treatment strategies. In particular, modulation ofphysiology of various, e.g., hematopoietic or lymphoid, cells will beachieved by appropriate methods for treatment using the compositionsprovided herein. See, e.g., Thomson (1994; ed.) The Cytokine Handbook(2d ed.) Academic Press, San Diego; Metcalf and Nicola (1995) TheHematopoietic Colony Stimulating Factors Cambridge University Press; andAggarwal and Gutterman (1991) Human Cytokines Blackwell Pub.

For example, a disease or disorder associated with abnormal expressionor abnormal signaling by an IL-B30 should be a likely target for anagonist or antagonist. The new cytokine should play a role in regulationor development of hematopoietic cells, e.g., lymphoid cells, whichaffect immunological responses, e.g., inflammation and/or autoimmunedisorders. Alternatively, it may affect vascular physiology ordevelopment, or neuronal effects.

In particular, the cytokine should mediate, in various contexts,cytokine synthesis by the cells, proliferation, etc. Antagonists ofIL-B30, such as mutein variants of a naturally occurring form of IL-B30or blocking antibodies, may provide a selective and powerful way toblock immune responses, e.g., in situations as inflammatory orautoimmune responses. See also Samter, et al. (eds.) ImmunologicalDiseases vols. 1 and 2, Little, Brown and Co.

In addition, certain combination compositions would be useful, e.g.,with other modulators of inflammation. Such other molecules may includesteroids, other versions of IL-6 and/or G-CSF, including speciesvariants, or viral homologs, and their respective antagonists.

Various abnormal conditions are known in each of the cell types shown toproduce IL-B30 mRNA by Northern blot analysis. See Berkow (ed.) TheMerck Manual of Diagnosis and Therapy, Merck & Co., Rahway, N.J.; Thorn,et al. Harrison's Principles of Internal Medicine, McGraw-Hill, N.Y.;and Weatherall, et al. (eds.) Oxford Textbook of Medicine, OxfordUniversity Press, Oxford. Many other medical conditions and diseasesinvolve activation by macrophages or monocytes, and many of these willbe responsive to treatment by an agonist or antagonist provided herein.See, e.g., Stites and Terr (eds.; 1991) Basic and Clinical ImmunologyAppleton and Lange, Norwalk, Conn.; and Samter, et al. (eds.)Immunological Diseases Little, Brown and Co. These problems should besusceptible to prevention or treatment using compositions providedherein. The pancreatic islet localization suggests a possible relevanceto diabetes.

IL-B30, antagonists, antibodies, etc., can be purified and thenadministered to a patient, veterinary or human. These reagents can becombined for therapeutic use with additional active or inertingredients, e.g., in conventional pharmaceutically acceptable carriersor diluents, e.g., immunogenic adjuvants, along with physiologicallyinnocuous stabilizers, excipients, or preservatives. These combinationscan be sterile filtered and placed into dosage forms as bylyophilization in dosage vials or storage in stabilized aqueouspreparations. This invention also contemplates use of antibodies orbinding fragments thereof, including forms which are not complementbinding.

Drug screening using IL-B30 or fragments thereof can be performed toidentify compounds having binding affinity to or other relevantbiological effects on IL-B30 functions, including isolation ofassociated components. Subsequent biological assays can then be utilizedto determine if the compound has intrinsic stimulating activity and istherefore a blocker or antagonist in that it blocks the activity of thecytokine. Likewise, a compound having intrinsic stimulating activity canactivate the signal pathway and is thus an agonist in that it simulatesthe activity of IL-B30. This invention further contemplates thetherapeutic use of blocking antibodies to IL-B30 as antagonists and ofstimulatory antibodies as agonists. This approach should be particularlyuseful with other IL-B30 species variants.

The quantities of reagents necessary for effective therapy will dependupon many different factors, including means of administration, targetsite, physiological state of the patient, and other medicantsadministered. Thus, treatment dosages should be titrated to optimizesafety and efficacy. Typically, dosages used in vitro may provide usefulguidance in the amounts useful for in situ administration of thesereagents. Animal testing of effective doses for treatment of particulardisorders will provide further predictive indication of human dosage.Various considerations are described, e.g., in Gilman, et al. (eds.1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics,8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences, 17thed. (1990), Mack Publishing Co., Easton, Pa. Methods for administrationare discussed therein and below, e.g., for oral, intravenous,intraperitoneal, or intramuscular administration, transdermal diffusion,and others. Pharmaceutically acceptable carriers will include water,saline, buffers, and other compounds described, e.g., in the MerckIndex, Merck & Co., Rahway, N.J. Dosage ranges would ordinarily beexpected to be in amounts lower than 1 mM concentrations, typically lessthan about 10 μM concentrations, usually less than about 100 nM,preferably less than about 10 μM (picomolar), and most preferably lessthan about 1 fM (femtomolar), with an appropriate carrier. Slow releaseformulations, or a slow release apparatus will often be utilized forcontinuous or long term administration. See, e.g., Langer (1990) Science249:1527-1533.

IL-B30, fragments thereof, and antibodies to it or its fragments,antagonists, and agonists, may be administered directly to the host tobe treated or, depending on the size of the compounds, it may bedesirable to conjugate them to carrier proteins such as ovalbumin orserum albumin prior to their administration. Therapeutic formulationsmay be administered in many conventional dosage formulations. While itis possible for the active ingredient to be administered alone, it ispreferable to present it as a pharmaceutical formulation. Formulationstypically comprise at least one active ingredient, as defined above,together with one or more acceptable carriers thereof. Each carriershould be both pharmaceutically and physiologically acceptable in thesense of being compatible with the other ingredients and not injuriousto the patient. Formulations include those suitable for oral, rectal,nasal, topical, or parenteral (including subcutaneous, intramuscular,intravenous and intradermal) administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethods well known in the art of pharmacy. See, e.g., Gilman, et al.(eds. 1990) Goodman and Gilman's: The Pharmacological Bases ofTherapeutics, 8th Ed., Pergamon Press; and Remington's PharmaceuticalSciences, 17th ed. (1990), Mack Publishing Co., Easton, Pa.; Avis, etal. (eds. 1993) Pharmaceutical Dosage Forms: Parenteral Medications,Dekker, New York; Lieberman, et al. (eds. 1990) Pharmaceutical DosageForms: Tablets, Dekker, New York; and Lieberman, et al. (eds. 1990)Pharmaceutical Dosage Forms: Disperse Systems, Dekker, New York. Thetherapy of this invention may be combined with or used in associationwith other agents, e.g., other cytokines, including IL-6 or G-CSF, ortheir respective antagonists.

Both naturally occurring and recombinant forms of the IL-B30s of thisinvention are particularly useful in kits and assay methods which arecapable of screening compounds for binding activity to the proteins.Several methods of automating assays have been developed in recent yearsso as to permit screening of tens of thousands of compounds in a shortperiod. See, e.g., Fodor, et al. (1991) Science 251:767-773, whichdescribes means for testing of binding affinity by a plurality ofdefined polymers synthesized on a solid substrate. The development ofsuitable assays can be greatly facilitated by the availability of largeamounts of purified, soluble IL-B30 as provided by this invention.

Other methods can be used to determine the critical residues inIL-B30-IL-B30 receptor interactions. Mutational analysis can beperformed, e.g., see Somoza, et al. (1993) J. Exptl. Med. 178:549-558,to determine specific residues critical in the interaction and/orsignaling. PHD (Rost and Sander (1994) Proteins 19:55-72) and DSC (Kingand Sternberg (1996) Protein Sci. 5:2298-2310) can provide secondarystructure predictions of α-helix (H), β-strand (E), or coil (L). HelicesA and D are most important in receptor interaction, with the D helix themore important region. Helix A would run in the human from about pro(7)to his(27), while helix D would run from about trp(135) to gly(162).Surface exposed residues would affect receptor binding, while embeddedresidues would affect general structure. Predicted residues ofparticular importance would likely correspond to arg(146), ser(147),gln(149), ala(150), ala(153), val(154), ala(156), arg(157), ala(160),and his(161).

For example, antagonists can normally be found once the antigen has beenstructurally defined, e.g., by tertiary structure data. Testing ofpotential interacting analogs is now possible upon the development ofhighly automated assay methods using a purified IL-B30. In particular,new agonists and antagonists will be discovered by using screeningtechniques described herein. Of particular importance are compoundsfound to have a combined binding affinity for a spectrum of IL-B30molecules, e.g., compounds which can serve as antagonists for speciesvariants of IL-B30.

One method of drug screening utilizes eukaryotic or prokaryotic hostcells which are stably transformed with recombinant DNA moleculesexpressing an IL-B30. Cells may be isolated which express an IL-B30 inisolation from other molecules. Such cells, either in viable or fixedform, can be used for standard binding partner binding assays. See also,Parce, et al. (1989) Science 246:243-247; and Owicki, et al. (1990)Proc. Nat'l Acad. Sci. USA 87:4007-4011, which describe sensitivemethods to detect cellular responses.

Another technique for drug screening involves an approach which provideshigh throughput screening for compounds having suitable binding affinityto an IL-B30 and is described in detail in Geysen, European PatentApplication 84/03564, published on Sep. 13, 1984. First, large numbersof different small peptide test compounds are synthesized on a solidsubstrate, e.g., plastic pins or some other appropriate surface, seeFodor, et al. (1991). Then all the pins are reacted with solubilized,unpurified or solubilized, purified IL-B30, and washed. The next stepinvolves detecting bound IL-B30.

Rational drug design may also be based upon structural studies of themolecular shapes of the IL-B30 and other effectors or analogs. Effectorsmay be other proteins which mediate other functions in response tobinding, or other proteins which normally interact with IL-B30, e.g., areceptor. One means for determining which sites interact with specificother proteins is a physical structure determination, e.g., x-raycrystallography or 2 dimensional NMR techniques. These will provideguidance as to which amino acid residues form molecular contact regions,as modeled, e.g., against other cytokine-receptor models. For a detaileddescription of protein structural determination, see, e.g., Blundell andJohnson (1976) Protein Crystallography, Academic Press, New York.

IX. Kits

This invention also contemplates use of IL-B30 proteins, fragmentsthereof, peptides, and their fusion products in a variety of diagnostickits and methods for detecting the presence of another IL-B30 or bindingpartner. Typically the kit will have a compartment containing either adefined IL-B30 peptide or gene segment or a reagent which recognizes oneor the other, e.g., IL-B30 fragments or antibodies.

A kit for determining the binding affinity of a test compound to anIL-B30 would typically comprise a test compound; a labeled compound, forexample a binding partner or antibody having known binding affinity forIL-B30; a source of IL-B30 (naturally occurring or recombinant); and ameans for separating bound from free labeled compound, such as a solidphase for immobilizing the molecule. Once compounds are screened, thosehaving suitable binding affinity to the antigen can be evaluated insuitable biological assays, as are well known in the art, to determinewhether they act as agonists or antagonists to the IL-B30 signalingpathway. The availability of recombinant IL-B30 polypeptides alsoprovide well defined standards for calibrating such assays.

A preferred kit for determining the concentration of, e.g., an IL-B30 ina sample would typically comprise a labeled compound, e.g., bindingpartner or antibody, having known binding affinity for the antigen, asource of cytokine (naturally occurring or recombinant) and a means forseparating the bound from free labeled compound, e.g., a solid phase forimmobilizing the IL-B30. Compartments containing reagents, andinstructions, will normally be provided.

Antibodies, including antigen binding fragments, specific for the IL-B30or fragments are useful in diagnostic applications to detect thepresence of elevated levels of IL-B30 and/or its fragments. Suchdiagnostic assays can employ lysates, live cells, fixed cells,immunofluorescence, cell cultures, body fluids, and further can involvethe detection of antigens related to the antigen in serum, or the like.Diagnostic assays may be homogeneous (without a separation step betweenfree reagent and antigen-binding partner complex) or heterogeneous (witha separation step). Various commercial assays exist, such asradioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA),enzyme immunoassay (EIA), enzyme-multiplied immunoassay technique(EMIT), substrate-labeled fluorescent immunoassay (SLFIA), and the like.See, e.g., Van Vunakis, et al. (1980) Meth Enzymol. 70:1-525; Harlow andLane (1980) Antibodies: A Laboratory Manual, CSH Press, NY; and Coligan,et al. (eds. 1993) Current Protocols in Immunology, Greene and Wiley,NY.

Anti-idiotypic antibodies may have similar use to diagnose presence ofantibodies against an IL-B30, as such may be diagnostic of variousabnormal states. For example, overproduction of IL-B30 may result inproduction of various immunological reactions which may be diagnostic ofabnormal physiological states, particularly in proliferative cellconditions such as cancer or abnormal activation or differentiation.Moreover, the distribution pattern available provides information thatthe cytokine is expressed in pancreatic islets, suggesting thepossibility that the cytokine may be involved in function of that organ,e.g., in a diabetes relevant medical condition.

Frequently, the reagents for diagnostic assays are supplied in kits, soas to optimize the sensitivity of the assay. For the subject invention,depending upon the nature of the assay, the protocol, and the label,either labeled or unlabeled antibody or binding partner, or labeledIL-B30 is provided. This is usually in conjunction with other additives,such as buffers, stabilizers, materials necessary for signal productionsuch as substrates for enzymes, and the like. Preferably, the kit willalso contain instructions for proper use and disposal of the contentsafter use. Typically the kit has compartments for each useful reagent.Desirably, the reagents are provided as a dry lyophilized powder, wherethe reagents may be reconstituted in an aqueous medium providingappropriate concentrations of reagents for performing the assay.

Many of the aforementioned constituents of the drug screening and thediagnostic assays may be used without modification or may be modified ina variety of ways. For example, labeling may be achieved by covalentlyor non-covalently joining a moiety which directly or indirectly providesa detectable signal. In any of these assays, the binding partner, testcompound, IL-B30, or antibodies thereto can be labeled either directlyor indirectly. Possibilities for direct labeling include label groups:radiolabels such as ¹²⁵I, enzymes (U.S. Pat. No. 3,645,090) such asperoxidase and alkaline phosphatase, and fluorescent labels (U.S. Pat.No. 3,940,475) capable of monitoring the change in fluorescenceintensity, wavelength shift, or fluorescence polarization. Possibilitiesfor indirect labeling include biotinylation of one constituent followedby binding to avidin coupled to one of the above label groups.

There are also numerous methods of separating the bound from the freeIL-B30, or alternatively the bound from the free test compound. TheIL-B30 can be immobilized on various matrixes followed by washing.Suitable matrixes include plastic such as an ELISA plate, filters, andbeads. See, e.g., Coligan, et al. (eds. 1993) Current Protocols inImmunology, Vol. 1, Chapter 2, Greene and Wiley, NY. Other suitableseparation techniques include, without limitation, the fluoresceinantibody magnetizable particle method described in Rattle, et al. (1984)Clin. Chem. 30:1457-1461, and the double antibody magnetic particleseparation as described in U.S. Pat. No. 4,659,678.

Methods for linking proteins or their fragments to the various labelshave been extensively reported in the literature and do not requiredetailed discussion here. Many of the techniques involve the use ofactivated carboxyl groups either through the use of carbodiimide oractive esters to form peptide bonds, the formation of thioethers byreaction of a mercapto group with an activated halogen such aschloroacetyl, or an activated olefin such as maleimide, for linkage, orthe like. Fusion proteins will also find use in these applications.

Another diagnostic aspect of this invention involves use ofoligonucleotide or polynucleotide sequences taken from the sequence ofan IL-B30. These sequences can be used as probes for detecting levels ofthe IL-B30 message in samples from patients suspected of having anabnormal condition, e.g., inflammatory or autoimmune. Since the cytokinemay be a marker or mediator for activation, it may be useful todetermine the numbers of activated cells to determine, e.g., whenadditional therapy may be called for, e.g., in a preventative fashionbefore the effects become and progress to significance. The preparationof both RNA and DNA nucleotide sequences, the labeling of the sequences,and the preferred size of the sequences has received ample descriptionand discussion in the literature. See, e.g., Langer-Safer, et al. (1982)Proc. Nat'l. Acad. Sci. 79:4381-4385; Caskey (1987) Science 236:962-967;and Wilchek et al. (1988) Anal. Biochem. 171:1-32.

Diagnostic kits which also test for the qualitative or quantitativeexpression of other molecules are also contemplated. Diagnosis orprognosis may depend on the combination of multiple indications used asmarkers. Thus, kits may test for combinations of markers. See, e.g.,Viallet, et al. (1989) Progress in Growth Factor Res. 1:89-97. Otherkits may be used to evaluate other cell subsets.

X. Isolating a IL-B30 Receptor

Having isolated a ligand of a specific ligand-receptor interaction,methods exist for isolating the receptor. See, Gearing, et al. (1989)EMBO J. 8:3667-3676. For example, means to label the IL-B30 cytokinewithout interfering with the binding to its receptor can be determined.For example, an affinity label can be fused to either the amino- orcarboxyl-terminus of the ligand. Such label may be a FLAG epitope tag,or, e.g., an Ig or Fc domain. An expression library can be screened forspecific binding of the cytokine, e.g., by cell sorting, or otherscreening to detect subpopulations which express such a bindingcomponent. See, e.g., Ho, et al. (1993) Proc. Nat'l Acad. Sci. USA90:11267-11271; and Liu, et al. (1994) J. Immunol. 152:1821-29.Alternatively, a panning method may be used. See, e.g., Seed and Aruffo(1987) Proc. Nat'l Acad. Sci. USA 84:3365-3369.

Protein cross-linking techniques with label can be applied to isolatebinding partners of the IL-B30 cytokine. This would allow identificationof proteins which specifically interact with the cytokine, e.g., in aligand-receptor like manner.

Early experiments will be performed to determine whether the known IL-6or G-CSF receptor components are involved in response(s) to IL-B30. Itis also quite possible that these functional receptor complexes mayshare many or all components with an IL-B30 receptor complex, either aspecific receptor subunit or an accessory receptor subunit.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

EXAMPLES I. General Methods

Many of the standard methods below are described or referenced, e.g., inManiatis, et al. (1982) Molecular Cloning, A Laboratory Manual ColdSpring Harbor Laboratory, Cold Spring Harbor Press, NY; Sambrook, et al.(1989) Molecular Cloning: A Laboratory Manual (2d ed.) Vols. 1-3, CSHPress, NY; Ausubel, et al., Biology Greene Publishing Associates,Brooklyn, N.Y.; or Ausubel, et al. (1987 and Supplements) CurrentProtocols in Molecular Biology Wiley/Greene, NY; Innis, et al. (eds.1990) PCR Protocols: A Guide to Methods and Applications Academic Press,NY. Methods for protein purification include such methods as ammoniumsulfate precipitation, column chromatography, electrophoresis,centrifugation, crystallization, and others. See, e.g., Ausubel, et al.(1987 and periodic supplements); Deutscher (1990) “Guide to ProteinPurification,” Methods in Enzymology vol. 182, and other volumes in thisseries; Coligan, et al. (1995 and supplements) Current Protocols inProtein Science John Wiley and Sons, New York, N.Y.; P. Matsudaira (ed.1993) A Practical Guide to Protein and Peptide Purification forMicrosequencing, Academic Press, San Diego, Calif.; and manufacturer'sliterature on use of protein purification products, e.g., Pharmacia,Piscataway, N.J., or Bio-Rad, Richmond, Calif. Combination withrecombinant techniques allow fusion to appropriate segments (epitopetags), e.g., to a FLAG sequence or an equivalent which can be fused,e.g., via a protease-removable sequence. See, e.g., Hochuli (1989)Chemische Industrie 12:69-70; Hochuli (1990) “Purification ofRecombinant Proteins with Metal Chelate Absorbent” in Setlow (ed.)Genetic Engineering, Principle and Methods 12:87-98, Plenum Press, NY;and Crowe, et al. (1992) OIAexpress: The High Level Expression & ProteinPurification System QUIAGEN, Inc., Chatsworth, Calif.

Standard immunological techniques are described, e.g., in Hertzenberg,et al. (eds. 1996) Weir's Handbook of Experimental Immunology vols. 1-4,Blackwell Science; Coligan (1991) Current Protocols in ImmunologyWiley/Greene, NY; and Methods in Enzymology vols. 70, 73, 74, 84, 92,93, 108, 116, 121, 132, 150, 162, and 163. Cytokine assays aredescribed, e.g., in Thomson (ed. 1994) The Cytokine Handbook (2d ed.)Academic Press, San Diego; Metcalf and Nicola (1995) The HematopoieticColony Stimulating Factors Cambridge University Press; and Aggarwal andGutterman (1991) Human Cytokines Blackwell Pub.

Assays for vascular biological activities are well known in the art.They will cover angiogenic and angiostatic activities in tumor, or othertissues, e.g., arterial smooth muscle proliferation (see, e.g., Koyoma,et al. (1996) Cell 87:1069-1078), monocyte adhesion to vascularepithelium (see McEvoy, et al. (1997) J. Exp. Med. 185:2069-2077), etc.See also Ross (1993) Nature 362:801-809; Rekhter and Gordon (1995) Am.J. Pathol. 147:668-677; Thyberg, et al. (1990) Atherosclerosis10:966-990; and Gumbiner (1996) Cell 84:345-357.

Assays for neural cell biological activities are described, e.g., inWouterlood (ed. 1995) Neuroscience Protocols modules 10, Elsevier;Methods in Neurosciences Academic Press; and Neuromethods Humana Press,Totowa, N.J. Methodology of developmental systems is described, e.g., inMeisami (ed.) Handbook of Human Growth and Developmental Biology CRCPress; and Chrispeels (ed.) Molecular Techniques and Approaches inDevelopmental Biology Interscience.

FACS analyses are described in Melamed, et al. (1990) Flow Cytometry andSorting Wiley-Liss, Inc., New York, N.Y.; Shapiro (1988) Practical FlowCytometry Liss, New York, N.Y.; and Robinson, et al. (1993) Handbook ofFlow Cytometry Methods Wiley-Liss, New York, N.Y.

II. Cloning of Human IL-B30

The sequence of the gene is provided in Table 1. The sequence is derivedfrom a cDNA library made from melanocyte, fetal heart, and pregnantuterus. It is also found from a cDNA library sequence derived from apancreatic islet. These sequences allow preparation of PCR primers, orprobes, to determine cellular distribution of the gene. The sequencesallow isolation of genomic DNA which encode the message.

Using the probe or PCR primers, various tissues or cell types are probedto determine cellular distribution. PCR products are cloned using, e.g.,a TA cloning kit (Invitrogen). The resulting cDNA plasmids are sequencedfrom both termini on an automated sequencer (Applied Biosystems).

III. Cellular Expression of IL-B30

An appropriate probe or primers specific for cDNA encoding primateIL-B30 are prepared. Typically, the probe is labeled, e.g., by randompriming. The expression is probably in the cell types described, andperhaps also in pancreatic islets. Southern Analysis: DNA (5 μg) from aprimary amplified cDNA library was digested with appropriate restrictionenzymes to release the inserts, run on a 1% agarose gel and transferredto a nylon membrane (Schleicher and Schuell, Keene, N.H.).

Samples for human mRNA isolation include: peripheral blood mononuclearcells (monocytes, T cells, NK cells, granulocytes, B cells), resting(T100); peripheral blood mononuclear cells, activated with anti-CD3 for2, 6, 12 h pooled (T101); T cell, TH0 clone Mot 72, resting (T102); Tcell, TH0 clone Mot 72, activated with anti-CD28 and anti-CD3 for 3, 6,12 h pooled (T103); T cell, TH0 clone Mot 72, anergic treated withspecific peptide for 2, 7, 12 h pooled (T104); T cell, TH1 clone HY06,resting (T107); T cell, TH1 clone HY06, activated with anti-CD28 andanti-CD3 for 3, 6, 12 h pooled (T108); T cell, TH1 clone HY06, anergictreated with specific peptide for 2, 6, 12 h pooled (T109); T cell, TH2clone HY935, resting (T110); T cell, TH2 clone HY935, activated withanti-CD28 and anti-CD3 for 2, 7, 12 h pooled (T111); T cell tumor linesJurkat and Hut78, resting (T117); T cell clones, pooled AD130.2,Tc783.12, Tc783.13, Tc783.58, Tc782.69, resting (T118); T cell random γδT cell clones, resting (T119); CD28-T cell clone; Splenocytes, resting(B100); Splenocytes, activated with anti-CD40 and IL-4 (B101); B cellEBV lines pooled WT49, RSB, JY, CVIR, 721.221, RM3, HSY, resting (B102);B cell line JY, activated with PMA and ionomycin for 1, 6 h pooled(B103); NK 20 clones pooled, resting (K101); NK 20 clones pooled,activated with PMA and ionomycin for 6 h (K101); NKL clone, derived fromperipheral blood of LGL leukemia patient, IL-2 treated (K106);hematopoietic precursor line TF1, activated with PMA and ionomycin for1, 6 h pooled (C100); U937 premonocytic line, resting (M100); U937premonocytic line, activated with PMA and ionomycin for 1, 6 h pooled(M101); elutriated monocytes, activated with LPS, IFNγ, anti-IL-10 for1, 2, 6, 12, 24 h pooled (M102); elutriated monocytes, activated withLPS, IFNγ, IL-10 for 1, 2, 6, 12, 24 h pooled (M103); elutriatedmonocytes, activated with LPS, IFNγ, anti-IL-10 for 4, 16 h pooled(M106); elutriated monocytes, activated with LPS, IFNγ, IL-10 for 4, 16h pooled (M107); elutriated monocytes, activated LPS for 1 h (M108);elutriated monocytes, activated LPS for 6 h (M109); DC 70% CD1a+, fromCD34+GM-CSF, TNFα 12 days, resting (D101); DC 70% CD1a+, fromCD34+GM-CSF, TNFα 12 days, activated with PMA and ionomycin for 1 hr(D102); DC 70% CD1a+, from CD34+GM-CSF, TNFα 12 days, activated with PMAand ionomycin for 6 hr (D103); DC 95% CD1a+, from CD34+GM-CSF, TNFα 12days FACS sorted, activated with PMA and ionomycin for 1, 6 h pooled(D104); DC 95% CD14+, ex CD34+GM-CSF, TNFα 12 days FACS sorted,activated with PMA and ionomycin 1, 6 hr pooled (D105); DC CD1a+CD86+,from CD34+GM-CSF, TNFα 12 days FACS sorted, activated with PMA andionomycin for 1, 6 h pooled (D106); DC from monocytes GM-CSF, IL-4 5days, resting (D107); DC from monocytes GM-CSF, IL-4 5 days, resting(D108); DC from monocytes GM-CSF, IL-4 5 days, activated LPS 4, 16 hpooled (D109); DC from monocytes GM-CSF, IL-4 5 days, activated TNFα,monocyte supe for 4, 16 h pooled (D110); epithelial cells, unstimulated;epithelial cells, IL-10 activated; lung fibroblast sarcoma line MRC5,activated with PMA and ionomycin for 1, 6 h pooled (C101); kidneyepithelial carcinoma cell line CHA, activated with PMA and ionomycin for1, 6 h pooled (C102). Expression of IL-B30 transcript was very high inelutriated monocytes, activated with LPS, IFNγ, anti-IL-10 for 4, 16 hpooled (M106); elutriated monocytes, activated with LPS, IFNγ,anti-IL-10 for 1, 2, 6, 12, 24 h pooled (M102); elutriated monocytes,activated LPS for 6 h (M109); and elutriated monocytes, activated LPSfor 1 h (M108). Expression was high in DC 95% CD1a+, from CD34+GM-CSF,TNFα 12 days FACS sorted, activated with PMA and ionomycin for 1, 6 hpooled (D104); and NK 20 clones pooled, activated with PMA and ionomycinfor 6 h (K101). Lesser expression was detected in DC 70% CD1a+, fromCD34+GM-CSF, TNFα 12 days, activated with PMA and ionomycin for 6 hr(D103); DC 70% CD1a+, from CD34+GM-CSF, TNFα12 days, activated with PMAand ionomycin for 1 hr (D102); T cell, TH1 clone HY06, anergic treatedwith specific peptide for 2, 6, 12 h pooled (T109); peripheral bloodmononuclear cells, activated with anti-CD3 for 2, 6, 12 h pooled (T101);T cell, TH0 clone Mot 72, activated with anti-CD28 and anti-CD3 for 3,6, 12 h pooled (T103); Splenocytes, activated with anti-CD40 and IL-4(B101); T cell, TH0 clone Mot 72, anergic treated with specific peptidefor 2, 7, 12 h pooled (T104); Splenocytes, resting (B101); T cell, TH1clone HY06, activated with anti-CD28 and anti-CD3 for 3, 6, 12 h pooled(T108); epithelial cells, IL-10 activated; elutriated monocytes,activated with LPS, IFNγ, IL-10 for 4, 16 h pooled (M107); and B cellline JY, activated with PMA and ionomycin for 1, 6 h pooled (B103).Detectable expression was observed in DC from monocytes GM-CSF, IL-4 5days, activated LPS 4, 16 h pooled (D109); T cell, TH0 clone Mot 72,resting (T102); peripheral blood mononuclear cells (monocytes, T cells,NK cells, granulocytes, B cells), resting (T100); T cells CD4+CD45RO-Tcells polarized 27 days in anti-CD28, IL-4, and anti IFN-γ, TH2polarized, activated with anti-CD3 and anti-CD28 4 h (T116); T cellclones, pooled AD130.2, Tc783.12, Tc783.13, Tc783.58, Tc782.69, resting(T118); U937 premonocytic line, resting (M100); hematopoietic precursorline TF1, activated with PMA and ionomycin for 1, 6 h pooled (C100); Tcell, TH2 clone HY935, activated with anti-CD28 and anti-CD3 for 2, 7,12 h pooled (T111); DC CD1a+CD86+, from CD34+GM-CSF, TNFα 12 days FACSsorted, activated with PMA and ionomycin for 1, 6 h pooled (D106);elutriated monocytes, activated with LPS, IFNγ, IL-10 for 1, 2, 6, 12,24 h pooled (M103); DC from monocytes GM-CSF, IL-4 5 days, activatedTNFα, monocyte supe for 4, 16 h pooled (D110); DC from monocytes GM-CSF,IL-4 5 days, resting (D108); U937 premonocytic line, activated with PMAand ionomycin for 1, 6 h pooled (M101); T cell random γδ T cell clones,resting (T119); and T cell, TH1 clone HY06, activated with anti-CD28 andanti-CD3 for 3, 6, 12 h pooled (T108). No signal was detected in theother samples.

In summary, the distribution shows IL-B30 elevated in activatedmacrophages, suggesting a role in inflammation; activated Th1 cells,suggesting a regulation or effector role in T helper subsets,particularly Th1 immune responses; and activated dendritic cells,suggesting a role in antigen presentation or germinal center T or B cellinteractions with DC.

Samples for mouse mRNA isolation include: resting mouse fibroblastic Lcell line (C200); Braf:ER (Braf fusion to estrogen receptor) transfectedcells, control (C201); Mel14+ naive T cells from spleen, resting (T209);Mel14+ naive T cells from spleen, stimulated with IFNγ, IL-12, and antiIL-4 to polarize to TH1 cells, exposed to IFNγ and IL-4 for 6, 12, 24 h,pooled (T210); Mel14+ naive T cells from spleen, stimulated with IL-4and anti IFNγ to polarize to Th2 cells, exposed to IL-4 and anti IFNγfor 6, 13, 24 h, pooled (T211); T cells, TH1 polarized (Mel14 bright,CD4+ cells from spleen, polarized for 7 days with IFN-γ and anti IL-4;T200); T cells, TH2 polarized (Mel14 bright, CD4+ cells from spleen,polarized for 7 days with IL-4 and anti-IFN-γ; T201); T cells, highlyTH1 polarized 3× from transgenic Balb/C (see Openshaw, et al. (1995) J.Exp. Med. 182:1357-1367; activated with anti-CD3 for 2, 6, 24 h pooled;T202); T cells, highly TH2 polarized 3× from transgenic Balb/C(activated with anti-CD3 for 2, 6, 24 h pooled (T203); T cells, highlyTH1 polarized 3× from transgenic C57 b1/6 (activated with anti-CD3 for2, 6, 24 h pooled; T212); T cells, highly TH2 polarized 3× fromtransgenic C57 b1/6 (activated with anti-CD3 for 2, 6, 24 h pooled;T213); T cells, highly TH1 polarized (naive CD4+ T cells from transgenicBalb/C, polarized 3× with IFNγ, IL-12, and anti-IL-4; stimulated withIGIF, IL-12, and anti IL-4 for 6, 12, 24 h, pooled); CD44−CD25+pre Tcells, sorted from thymus (T204); TH1 T cell clone D1.1, resting for 3weeks after last stimulation with antigen (T205); TH1 T cell clone D1.1,10 μg/ml ConA stimulated 15 h (T206); TH2 T cell clone CDC35, restingfor 3 weeks after last stimulation with antigen (T207); TH2 T cell cloneCDC35, 10 μg/ml ConA stimulated 15 h (T208); unstimulated B cell lineCH12 (B201); unstimulated mature B cell leukemia cell line A20 (B200);unstimulated large B cells from spleen (B202); B cells from totalspleen, LPS activated (B203); metrizamide enriched dendritic cells fromspleen, resting (D200); dendritic cells from bone marrow, resting(D201); unstimulated bone marrow derived dendritic cells depleted withanti B220, anti CD3, and anti Class II, cultured in GM-CSF and IL-4(D202); bone marrow derived dendritic cells depleted with anti B220,anti CD3, and anti Class II, cultured in GM-CSF and IL-4, stimulatedwith anti CD40 for 1, 5 d, pooled (D203); monocyte cell line RAW 264.7activated with LPS 4 h (M200); bone-marrow macrophages derived with GMand M-CSF (M201); bone-marrow macrophages derived with GM-CSF,stimulated with LPS, IFNγ, and IL-10 for 24 h (M205); bone-marrowmacrophages derived with GM-CSF, stimulated with LPS, IFNγ, and antiIL-10 for 24 h (M206); peritoneal macrophages (M207); macrophage cellline J774, resting (M202); macrophage cell line J774+LPS+anti-IL-10 at0.5, 1, 3, 6, 12 h pooled (M203); macrophage cell line J774+LPS+IL-10 at0.5, 1, 3, 5, 12 h pooled (M204); unstimulated mast cell lines MC-9 andMCP-12 (M208); immortalized endothelial cell line derived from brainmicrovascular endothelial cells, unstimulated (E200); immortalizedendothelial cell line derived from brain microvascular endothelialcells, stimulated overnight with TNFα (E201); immortalized endothelialcell line derived from brain microvascular endothelial cells, stimulatedovernight with TNFα (E202); immortalized endothelial cell line derivedfrom brain microvascular endothelial cells, stimulated overnight withTNFα and IL-10 (E203); total aorta from wt C57 b1/6 mouse; total aortafrom 5 month ApoE KO mouse (X207); total aorta from 12 month ApoE KOmouse (X207); wt thymus (O214); total thymus, rag-1 (O208); totalkidney, rag-1 (0209); total kidney, NZ B/W mouse; and total heart, rag-1(0202). High signal was detected in the monocyte cell line RAW 264.7activated with LPS 4 h (M200); T cells, highly TH1 polarized 3× fromtransgenic C57 b1/6 (activated with anti-CD3 for 2, 6, 24 h pooled;T212); and T cells, highly TH1 polarized (naive CD4+ T cells fromtransgenic Balb/C, polarized 3× with IFNγ, IL-12, and anti-IL-4;stimulated with IGIF, IL-12, and anti IL-4 for 6, 12, 24 h, pooled).Detectable signals were detected in T cells, highly TH1 polarized 3×from transgenic Balb/C (see Openshaw, et al. (1995) J. Exp. Med.182:1357-1367; activated with anti-CD3 for 2, 6, 24 h pooled; T202); Tcells, TH2 polarized (Mel14 bright, CD4+ cells from spleen, polarizedfor 7 days with IL-4 and anti-IFN-γ; T201); T cells, TH1 polarized(Mel14 bright, CD4+ cells from spleen, polarized for 7 days with IFN-γand anti IL-4; T200); macrophage cell line J774+LPS+anti-IL-10 at 0.5,1, 3, 6, 12 h pooled (M203); macrophage cell line J774, resting (M202);macrophage cell line J774+LPS+IL-10 at 0.5, 1, 3, 5, 12 h pooled (M204);immortalized endothelial cell line derived from brain microvascularendothelial cells, stimulated overnight with TNFα (E201); andbone-marrow macrophages derived with GM-CSF, stimulated with LPS, IFNγ,and anti IL-10 for 24 h (M206). Other samples showed no signal. Theexpression in the RAW 264.7 mouse monocyte cell line suggests a naturalsource for protein.

IV. Chromosome Mapping of IL-B30

An isolated cDNA encoding the IL-B30 is used. Chromosome mapping is astandard technique. See, e.g., BIOS Laboratories (New Haven, Conn.) andmethods for using a mouse somatic cell hybrid panel with PCR.Circumstantial evidence suggests that the mouse gene is localized onchromosome 10.

V. Purification of IL-B30 Protein

Multiple transfected cell lines are screened for one which expresses thecytokine at a high level compared with other cells. Various cell linesare screened and selected for their favorable properties in handling.Natural IL-B30 can be isolated from natural sources, or by expressionfrom a transformed cell using an appropriate expression vector.Purification of the expressed protein is achieved by standardprocedures, or may be combined with engineered means for effectivepurification at high efficiency from cell lysates or supernatants. FLAGor His₆ segments can be used for such purification features.Alternatively, affinity chromatography may be used with specificantibodies, see below.

Protein is produced in coli, insect cell, or mammalian expressionsystems, as desired.

VI. Isolation of Homologous IL-B30 Genes

The IL-B30 cDNA, or other species counterpart sequence, can be used as ahybridization probe to screen a library from a desired source, e.g., aprimate cell cDNA library. Many different species can be screened bothfor stringency necessary for easy hybridization, and for presence usinga probe. Appropriate hybridization conditions will be used to select forclones exhibiting specificity of cross hybridization.

Screening by hybridization using degenerate probes based upon thepeptide sequences will also allow isolation of appropriate clones.Alternatively, use of appropriate primers for PCR screening will yieldenrichment of appropriate nucleic acid clones.

Similar methods are applicable to isolate either species, polymorphic,or allelic variants. Species variants are isolated using cross-specieshybridization techniques based upon isolation of a full length isolateor fragment from one species as a probe.

Alternatively, antibodies raised against human IL-B30 will be used toscreen for cells which express cross-reactive proteins from anappropriate, e.g., cDNA library. The purified protein or definedpeptides are useful for generating antibodies by standard methods, asdescribed above. Synthetic peptides or purified protein are presented toan immune system to generate monoclonal or polyclonal antibodies. See,e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene; andHarlow and Lane (1989) Antibodies: A Laboratory Manual Cold SpringHarbor Press. The resulting antibodies are used for screening,purification, or diagnosis, as described.

VII. Preparation of Antibodies Specific for IL-B30

Synthetic peptides or purified protein are presented to an immune systemto generate monoclonal or polyclonal antibodies. See, e.g., Coligan(1991) Current Protocols in Immunology Wiley/Greene; and Harlow and Lane(1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press.Polyclonal serum, or hybridomas may be prepared. In appropriatesituations, the binding reagent is either labeled as described above,e.g., fluorescence or otherwise, or immobilized to a substrate forpanning methods. Immunoselection and related techniques are available toprepare selective reagents, as desired.

VIII. Evaluation of Breadth of Biological Functions

Biological activities of IL-B30 were tested based on the sequence andstructural homology between IL-B30 and IL-6 and G-CSF. Initially, assaysthat had shown biological activities of IL-6 or G-CSF are examined.

A. Effects on Proliferation of Cells

The effect on proliferation of various cell types are evaluated withvarious concentrations of cytokine. A dose response analysis isperformed, in combinations with the related cytokines IL-6, G-CSF, etc.

B. Effects on the Expression of Cell Surface Molecules On HumanMonocytes

Monocytes are purified by negative selection from peripheral bloodmononuclear cells of normal healthy donors. Briefly, 3×10⁸ ficoll bandedmononuclear cells are incubated on ice with a cocktail of monoclonalantibodies (Becton-Dickinson; Mountain View, Calif.) consisting, e.g.,of 200 μl of αCD2 (Leu-5A), 200 μl of αCD3 (Leu-4), 100 μl of αCD8 (Leu2a), 100 μl of αCD19 (Leu-12), 100 μl of αCD20 (Leu-16), 100 μl of αCD56(Leu-19), 100 μl of αCD67 (10M 67; Immunotech, Westbrook, Me.), andanti-glycophorin antibody (10F7MN, ATCC, Rockville, Md.). Antibody boundcells are washed and then incubated with sheep anti-mouse IgG coupledmagnetic beads (Dynal, Oslo, Norway) at a bead to cell ratio of 20:1.Antibody bound cells are separated from monocytes by application of amagnetic field. Subsequently, human monocytes are cultured in Yssel'smedium (Gemini Bioproducts, Calabasas, Calif.) containing 1% human ABserum in the absence or presence of IL-B30, IL-6, G-CSF or combinations.

Analyses of the expression of cell surface molecules can be performed bydirect immunofluorescence. For example, 2×10⁵ purified human monocytesare incubated in phosphate buffered saline (PBS) containing 1% humanserum on ice for 20 minutes. Cells are pelleted at 200×g. Cells areresuspended in 20 ml PE or FITC labeled mAb. Following an additional 20minute incubation on ice, cells are washed in PBS containing 1% humanserum followed by two washes in PBS alone. Cells are fixed in PBScontaining 1% paraformaldehyde and analyzed on FACScan flow cytometer(Becton Dickinson; Mountain View, Calif.). Exemplary mabs are used,e.g.: CD11b (anti-mac1), CD11c (a gp150/95), CD14 (Leu-M3), CD54 (Leu54), CD80 (anti-BB1/B7), HLA-DR (L243) from Becton-Dickinson and CD86(FUN 1; Pharmingen), CD64 (32.2; Medarex), CD40 (mAb89; Schering-PloughFrance).

C. Effects of IL-B30 on Cytokine Production by Human Monocytes

Human monocytes are isolated as described and cultured in Yssel's medium(Gemini Bioproducts, Calabasas, Calif.) containing 1% human AB serum inthe absence or presence of IL-B30 ( 1/100 dilution baculovirus expressedmaterial). In addition, monocytes are stimulated with LPS (E. coli0127:B8 Difco) in the absence or presence of IL-B30 and theconcentration of cytokines (IL-1β, IL-6, TNFα, GM-CSF, and IL-10) in thecell culture supernatant determined by ELISA.

For intracytoplasmic staining for cytokines, monocytes are cultured (1million/ml) in Yssel's medium in the absence or presence of IL-B30 andLPS (E. coli 0127:B8 Difco) and 10 mg/ml Brefeldin A (Epicentretechnologies Madison Wis.) for 12 hrs. Cells are washed in PBS andincubated in 2% formaldehyde/PBS solution for 20 minutes at RT.Subsequently cells are washed, resuspended in permeabilization buffer(0.5% saponin (Sigma) in PBS/BSA (0.5%)/Azide (1 mM)) and incubated for20 minutes at RT. Cells (2×10⁵) are centrifuged and resuspended in 20 mldirectly conjugated anti-cytokine mAbs diluted 1:10 in permeabilizationbuffer for 20 minutes at RT. The following antibodies can be used:IL-1α-PE (364-3B3-14); IL-6-PE (MQ2-13A5); TNFα-PE (MAbli); GM-CSF-PE(BVD2-21C11); and IL-12-PE (C11.5.14; Pharmingen San Diego, Calif.).Subsequently, cells are washed twice in permeabilization buffer and oncein PBS/BSA/Azide and analyzed on FACScan flow cytometer (BectonDickinson; Mountain View, Calif.).

D. Effects of IL-B30 on Proliferation of Human Peripheral BloodMononuclear Cells (PBMC).

Total PBMC are isolated from buffy coats of normal healthy donors bycentrifugation through ficoll-hypaque as described (Boyum, et al.). PBMCare cultured in 200 μl Yssel's medium (Gemini Bioproducts, Calabasas,Calif.) containing 1% human AB serum in 96 well plates (Falcon,Becton-Dickinson, N.J.) in the absence or presence of IL-B30. Cells arecultured in medium alone or in combination with 100 U/ml IL-2 (R&DSystems) for 120 hours. 3H-Thymidine (0.1 mCi) is added during the lastsix hours of culture and 3H-Thymidine incorporation determined by liquidscintillation counting.

The native, recombinant, and fusion proteins would be tested for agonistand antagonist activity in many other biological assay systems, e.g., onT-cells, B-cells, NK, macrophages, dendritic cells, hematopoieticprogenitors, etc. Because of the IL-6 and G-CSF structural relationship,assays related to those activities should be analyzed

IL-B30 is evaluated for agonist or antagonist activity on transfectedcells expressing IL-6 or G-CSF receptor and controls. See, e.g., Ho, etal. (1993) Proc. Nat'l Acad. Sci. USA 90, 11267-11271; Ho, et al. (1995)Mol. Cell. Biol. 15:5043-5053; and Liu, et al. (1994). J. Immunol.152:1821-1829.

IL-B30 is evaluated for effect in macrophage/dendritic cell activationand antigen presentation assays, T cell cytokine production andproliferation in response to antigen or allogeneic stimulus. See, e.g.,de Waal Malefyt et al. (1991) J. Exp. Med. 174:1209-1220; de WaalMalefyt et al. (1991) J. Exp. Med. 174:915-924; Fiorentino, et al.(1991) J. Immunol. 147, 3815-3822; Fiorentino, et al. (1991) J. Immunol.146:3444-3451; and Groux, et al. (1996) J. Exp. Med. 184:19-29.

IL-B30 will also be evaluated for effects on NK cell stimulation. Assaysmay be based, e.g., on Hsu, et al. (1992) Internat. Immunol. 4:563-569;and Schwarz, et al. (1994) J. Immunother. 16:95-104.

B cell growth and differentiation effects will be analyzed, e.g., by themethodology described, e.g., in Defrance, et al. (1992). J. Exp. Med.175:671-682; Rousset, et al. (1992) Proc. Nat'l Acad. Sci. USA89:1890-1893; including IgG2 and IgA2 switch factor assays. Note that,unlike COS7 supernatants, NIH3T3 and COP supernatants apparently do notinterfere with human B cell assays.

IX. Generation and Analysis of Genetically Altered Animals

Transgenic mice can be generated by standard methods.

Such animals are useful to determine the effects of deletion of thegene, in specific tissues, or completely throughout the organism. Suchmay provide interesting insight into development of the animal orparticular tissues in various stages. Moreover, the effect on variousresponses to biological stress can be evaluated. See, e.g., Hogan, etal. (1995) Manipulating the Mouse Embryo: A Laboratory Manual (2d ed.)Cold Spring Harbor Laboratory Press.

A transgenic mouse has been generated, and while the animal seems tosurvive birth, it fails to thrive, and typically dies within a fewweeks. The construct is based upon an actin promoter with a CMVenhancer, which should lead to broad and high expression. The mice, likeIL-6 transgenic mice, are runted. Moreover, they exhibit a bloatedabdomen, inflammation of the stomach and intestines, infiltration ofcells into the liver, and typically die before day 50. These mice do notbreed. A second subset of the transgenic mice have a less severephenotype, and attempts to breed them are taking place.

The genomic structure for the mouse IL-B30 has been determined. Astrategy for the production of IL-B30 knock-out mice has been developed,and constructs have been started.

All references cited herein are incorporated herein by reference to thesame extent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety for all purposes.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

1. An isolated or recombinant polynucleotide encoding an antigenicpolypeptide comprising: a) at least 17 contiguous amino acids from themature coding portion of SEQ ID NO: 2; b) at least 17 contiguous aminoacids from the mature coding portion of SEQ ID NO: 4; or c) at least 17contiguous amino acids from the mature coding portion of SEQ ID NO: 5.2. The polynucleotide of claim 1, encoding a mature polypeptide of: a)SEQ ID NO: 2; or b) SEQ ID NO:
 4. 3. The polynucleotide of claim 1,which hybridizes at 55° C., less than 500 mM salt, and 50% formamide to:a) the coding portions of SEQ ID NO: 1; or b) the coding portions of SEQID NO:
 3. 4. The polynucleotide of claim 3, comprising: a) at least 35contiguous nucleotides of the coding portion of SEQ ID NO: 1; or b) atleast 35 contiguous nucleotides of the coding portion of SEQ ID NO: 3.5. An expression vector comprising the polynucleotide of claim
 1. 6. Ahost cell containing the expression vector of claim 5, including aeukaryotic cell.
 7. A method of making an antigenic polypeptidecomprising expressing a recombinant polynucleotide of claim
 1. 8. Amethod for the detection of a polynucleotide of claim 1, comprisingcontacting said polynucleotide with a probe that hybridizes, understringent conditions, to at least 25 contiguous nucleotides of: a) thecoding portion of SEQ ID NO: 1; or b) the coding portion of SEQ ID NO:3; to form a duplex, wherein detection of said duplex indicates thepresence of said polynucleotide.
 9. A kit for the detection of apolynucleotide of claim 1, comprising a compartment containing a probethat hybridizes, under stringent hybridization conditions, to at least17 contiguous nucleotides of a polynucleotide of claim 1 to form aduplex.
 10. The kit of claim 9, wherein said probe is detectablylabeled.
 11. A binding compound comprising an antibody binding sitewhich specifically binds to: a) at least 17 contiguous amino acids fromSEQ ID NO: 2; b) at least 17 contiguous amino acids from SEQ ID NO: 4;or c) at least 17 contiguous amino acids from SEQ ID NO:
 5. 12. Thebinding compound of claim 11, wherein: a) said antibody binding siteis: 1) specifically immunoreactive with a polypeptide of SEQ ID NO: 2;2) specifically immunoreactive with a polypeptide of SEQ ID NO: 4; 3)specifically immunoreactive with a polypeptide of SEQ ID NO: 5; 4)raised against a purified or recombinantly produced human IL-B30protein; 5) raised against a purified or recombinantly produced mouseIL-B30; 6) in a monoclonal antibody, Fab, or F(ab)₂; or b) said bindingcompound is: 1) an antibody molecule; 2) a polyclonal antiserum; 3)detectably labeled; 4) sterile; or 5) in a buffered composition.
 13. Amethod using the binding compound of claim 11, comprising contactingsaid binding compound with a biological sample comprising an antigen,wherein said contacting results in formation of a bindingcompound:antigen complex.
 14. The method of claim 13, wherein saidbiological sample is from a human, and wherein said binding compound isan antibody.
 15. A detection kit comprising said binding compound ofclaim 12, and: a) instructional material for the use of said bindingcompound for said detection; or b) a compartment providing segregationof said binding compound.
 16. A substantially pure or isolated antigenicpolypeptide, which binds to said binding composition of claim 11, andfurther comprises: a) at least 17 contiguous amino acids from SEQ ID NO:2; b) at least 17 contiguous amino acids from SEQ ID NO: 4; or c) atleast 17 contiguous amino acids from SEQ ID NO:
 5. 17. The polypeptideof claim 16, which: a) comprises at least a fragment of at least 25contiguous amino acid residues from a primate IL-B30 protein; b)comprises at least a fragment of at least 25 contiguous amino acidresidues from a rodent IL-B30 protein; c) is a soluble polypeptide; d)is detectably labeled; e) is in a sterile composition; f) is in abuffered composition; g) binds to a cell surface receptor; h) isrecombinantly produced; or i) has a naturally occurring polypeptidesequence.
 18. The polypeptide of claim 17, which: a) comprises at least17 contiguous amino acids of SEQ ID NO: 2; b) comprises at least 17contiguous amino acids of SEQ ID NO: 4; or c) comprises at least 17contiguous amino acids of SEQ ID NO:
 5. 19. A method of modulatingphysiology or development of a cell or tissue culture cells comprisingcontacting said cell with an agonist or antagonist of a mammalianIL-B30.
 20. The method of claim 19, wherein: a) said contacting is incombination with an agonist or antagonist of G-CSF and/or IL-6; or b)said contacting is with an antagonist, including binding compositioncomprising an antibody binding site which specifically binds an IL-B30.